Impaired “outside-in” integrin α_IIbβ₃ signaling and thrombus stability in TSSC6-deficient mice

Tumor-suppressing subchromosomal transferable fragment cDNA 6 (TSSC6), also previously known as Pan hematopoietic expression (Phemx), is a tetraspanin superfamily member (transmembrane 4 superfamily).^1,2  TSSC6 contains 4 highly conserved hydrophobic transmembrane domains, 2 extracellular loops, and intracellular N- and C-terminal domains. Its C-terminal cytoplasmic domain is relatively large (33 and 99 amino acids in mouse and human, respectively) compared to other tetraspanin superfamily members. TSSC6 is specifically expressed in hematopoietic organs and tissues where it may play a role in hematopoietic-cell function. Human TSSC6 transcripts have been detected in K562 cells stimulated with phorbol myristate acetate (PMA) to induce differentiation into megakaryocyte lineage.^1,3  This finding suggests that TSSC6 protein is likely to be present in platelets and where it may have a functional role.

A hallmark feature of tetraspanin superfamily members is the presence of 4 highly conserved cysteine motifs in the major extracellular domain 2, including CCG, PXSC, and EGC.⁴  Tetraspanins form physical multimolecular complexes with other transmembrane proteins and transmembrane receptors including integrins that constitute a network termed the tetraspanin web. Several studies have highlighted associations between tetraspanins and integrins, and the best characterized of these include integrin α₃β₁ with CD151, α₆β₁ and α₆β4 with CD151, and α₄β₁ with CD81.⁵  As yet, no biochemical or physical associations of TSSC6 with integrins, including platelet integrin α_IIbβ₃, have been defined.

There have been several major tetraspanins reported in platelets, including CD9, CD151, TSSC6, and CD63. Of these, CD9 and CD151 are present on the platelet surface and are also associated with the inner surface of alpha granules, along with their molecular partner, integrin α_IIbβ₃ and its soluble ligand fibrinogen.⁶  In contrast, CD63 is present on the membrane of dense granules and lysosomes and is relocated to the platelet surface following platelet activation and exocytosis.⁷  CD63 has been demonstrated to be physically associated with a multimolecular complex of CD9: integrin α_IIbβ₃ on the surface of activated platelets.⁸  The intracellular localization of TSSC6 in platelets is presently unknown.

In platelets, CD9 has been functionally linked with the low-affinity IgG receptor, FcγRIIa,^9,10  although others suggest that CD9 may regulate platelet function independently of FcγRIIa.¹¹  CD151 functions independently of platelet FcγRIIa and regulates the “outside-in” signaling properties of integrin α_IIbβ₃ in murine platelets that are devoid of FcγRIIa.^12,13  Recently, CD63 was reported to modulate human platelet spreading on immobilized fibrinogen in an integrin α_IIbβ₃-dependent manner.⁷  These studies demonstrate that the formation of tetraspanin multimolecular complexes with integrin α_IIbβ₃ in platelets is of functional importance.

In this study, we report that the hematopoietic-specific tetraspanin superfamily member, TSSC6, is expressed both on the cell surface and in intracellular pools of murine platelets. In this context, TSSC6 is physically associated with the integrin α_IIbβ₃ complex in platelets and has a direct role in regulating “outside-in” integrin α_IIbβ₃-mediated signaling in murine platelets. In addition, TSSC6 functions as a molecular facilitator to stabilize platelet aggregates in high-shear thrombosis following vascular injury in vivo.

TSSC6-deficient mice were generated in the laboratory of our authors (L.R., M.D.W.) and maintained on a C57BL/6 background.¹⁴  Four- to 8-week-old wild-type (WT) and TSSC6 null mice were used in this study. Experimental procedures were approved by the Austin Health Animal Ethics Committee.

Anti–mouse integrin β₃ mAb, FITC-conjugated anti–mouse P-selectin mAb, FITC-conjugated anti–mouse CD3 mAb, and phycoerythrin (PE)–conjugated antirat Ab were purchased from BD Pharmingen (San Diego, CA). Anti–human integrin complex–specific α_IIbβ₃ Ab, P2, was obtained from Beckman Coulter (Gladesville, New South Wales). FITC-conjugated anti–mouse CD44 mAb was obtained from Beckman Coulter (Brea, CA), while rat anti–mouse CD9 mAb and JON/A-PE mAb were from Emfret Analytics (Wurzburg, Germany). Anti–mouse TSSC6 monoclonal antibodies (15G3 and 14A6) were generated by immunizing Wistar rats with FDC-P1 and DP16 cell lines. Primary screening of hybridoma supernatants were performed by flow cytometry of a mixture of COS-7 cells engineered to express the FLAG epitope in EC1 (COS-7 TSSC6/EC1 FLAG) and parental COS-7 cells.¹⁵  Selected supernatants were rescreened against COS-7 TSSC6/EC1 FLAG and panel of murine hematopoietic-cell lines. Polyclonal anti–human TSSC6 (Phemx) Ab was obtained from Abcam (Cambridge, United Kingdom). FITC-conjugated fibrinogen was generated by incubating 20 mg human fibrinogen with 5 mg FITC (Sigma Chemical, St Louis, MO) in 0.15 M carbonate buffer pH 9.0 for 1 hour at room temperature. Labeled fibrinogen was then purified by separation over a PD10 column (Amersham Pharmacia, Piscataway, NJ).

Eight hundred microliters of whole blood was collected by cardiac puncture of the inferior vena cava of each mouse under halothane anesthesia. The whole blood was anticoagulated in 3.8% (wt/vol) trisodium citrate in a ratio of 1 part anticoagulant to 9 parts blood. For washed platelet assays, 50 ng/mL prostaglandin E₁ (PGE₁) (Sigma Chemical) was included to minimize platelet activation. Platelets were obtained from platelet-rich plasma and washed in Ringers citrate dextrose buffer (RCD) pH 6.5. Platelet counts were normalized between wild-type and TSSC6 null platelets before functional assays were performed.

Tail-bleeding times and clot retraction were performed as previously described.^13,16 

Platelet aggregation was stimulated by 2.5 to 20 μg/mL collagen, 2.5 to 10 μM adenosine diphosphate (ADP), 5 to 20 μg/mL calcium ionophore, 125 to 500 μM protease-activated receptor (PAR-4) agonist peptide, 2.5 to 20 μg/mL collagen-related peptide (CRP), and monitored in a 4-channel Chronolog aggregometer (Chrono-log, Havertown, PA). Fibrinogen was added at a final concentration of 100 μg/mL.

Washed platelets were resuspended in RCD buffer pH 6.5 and stimulated with a range of agonists as indicated. Platelets (100 × 10⁹/L) were incubated with either FITC-conjugated rat anti–mouse P-selectin (10 μg/mL), FITC-conjugated human fibrinogen, or PE-conjugated JON/A antibody for 1 hour at 37°C. Samples were washed in RCD pH 6.5 containing 0.2% (wt/vol) bovine serum albumin and then analyzed immediately. For quantitative flow cytometry, QuantiBrite PE beads at 4 different levels of molecules/bead were used to derive a calibration curve (Becton Dickinson, San Jose, CA). For all measurements, a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) was used.

Platelet adhesion to fibrinogen was performed as previously described.¹³  Briefly, coverslips were coated with 100 μg/mL fibrinogen, and washed platelets (100 × 10⁹/L) were added. Attached platelets were visualized using differential interference contrast (DIC) microscopy (×63 DIC oil objective), and the numbers of attached platelets per high-power field were counted. Similarly, for platelet-spreading experiments, unlabeled platelets were visualized using DIC microscopy (×63 DIC oil objective). Spread platelets were differentiated from nonspread platelets counted per high-power field according to the criteria of Goodman.¹⁷  Using this criteria, spreading platelets (S) were defined as a hyaloplasm spread between pseudopodia or fully spread platelets (FS) as hyaloplasm extensively spread with no distinct pseudopodia. As mouse platelets undergo a more limited degree of spreading on fibrinogen than human platelets, the S forms of platelets were more common than FS.

Wild-type or TSSC6-deficient C57BL/6 mice (4-5 weeks old) were anesthetized with a ketamine/xyzaline (200:10 mg/kg) mixture, and the mesentery was exteriorized through a midline abdominal incision. A catheter was inserted into the jugular vein to deliver rhodamine dye and anesthetic as required. Mesenteric arterioles (60-100 μm) were visualized with a Zeiss Axiovert 135 microscope (Zeiss, Oberkochen, Germany), captured with a Dage MTI camera (Dage MTI, Michigan City, IN), and video recorded using Streamline software using a Dell computer. A filter paper strip of 1 × 4 mm was dipped in a 462 mM (7.5% wt/vol) FeCl₃ solution for 3 seconds and then applied to a 2- to 5-mm length of arteriole for 4 minutes and then removed.¹⁸  Vessels were monitored for 30 minutes after injury or until full occlusion (blood flow stopped). Parameters assessed were (1) the time required for initial thrombus formation of diameter more than 20 μm; (2) the number of thrombi of diameter more than 20 μm; (3) clot occlusion time of vessel that involves the time for the blood to stop flowing; (4) the increase in area and diameter over 1 minute for forming thrombi more than 20 μm; (5) the time the blood clot was stable in the vessel (seconds); (6) duration of instability of blood clot in occluded vessel over time (seconds) (if observed); and (7) the number of emboli observed after vessel has clotted (termed secondary stability).

Washed platelets were diluted using the Unopette microcollection system according to the manufacturer's instructions (Becton Dickinson, Franklin Lakes, NJ). Platelet counts were performed using a hemocytometer.

Data are presented as mean ± SEM. Statistical significance was assessed by unpaired Student t test and the Mann-Whitney test as indicated using GraphPad Prism statistical software version 4 (GraphPad, San Diego, CA). P values less than .05 were considered significant.

Reverse transcriptase–polymerase chain reaction studies of TSSC6 transcripts in tissues and cells have demonstrated that TSSC6 is a hematopoietic-specific tetraspanin.¹  To determine the presence of TSSC6 protein expressed on the surface of resting platelets and to determine if this expression is up-regulated by intracellular granule release, we examined the binding of specific monoclonal anti–murine TSSC6 antibodies (14A6) to either resting or thrombin-stimulated murine platelets, monitored by flow cytometry. As shown in Figure 1A, a 2-fold increase in fluorescence intensity was observed in resting murine platelets using specific monoclonal antibodies directed against TSSC6 (n = 3). This was further increased another 2-fold by thrombin stimulation under conditions of alpha granule release, as shown by P-selectin exposure (Figure 1B). This increase in TSSC6 expression plateaued around 2 U/mL thrombin (n = 3). As a negative control, TSSC6 knockout platelets were included and showed minimal background with the TSSC6 monoclonal antibody (Figure 1B). In addition, using quantitative flow cytometry to determine relative molecules per cell for TSSC6 versus CD9 on murine platelets, we determined that CD9 was expressed 5-fold higher in expression than TSSC6 on murine platelets (8930 ± 1218 molecules/cell versus 2281 ± 214 molecules/cell). These results indicate that TSSC6 is expressed on the surface of resting murine platelets and is found in an intracellular pool in murine platelets.

To investigate a physical association of integrin α_IIbβ₃ complex with TSSC6 in human platelets, immunoprecipitation studies were performed using antibodies directed against isotype control mAb, normal mouse IgG₁, platelet glycoprotein platelet-endothelial-cell adhesion molecule-1 (PECAM-1), GPIb-IX-V complex, and integrin α_IIbβ₃ complex–specific mAb, P2, in the presence of either 1% (vol/vol) Triton X-100 or with 1% (vol/vol) Brij-96, 1% (vol/vol) Thesit, and 1% (vol/vol) CHAPS 3-([(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate). Following immunoprecipitation, protein complexes were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and the presence of TSSC6 detected by Western blotting. As shown in Figure 1C, TSSC6 was detectable in integrin α_IIbβ₃ (P2) immunoprecipitates, but not normal mouse IgG₁, GPIb-IX-V complex, and PECAM-1 immunoprecipitates, under all detergent conditions including 1% (vol/vol) Triton X-100, 1% (vol/vol) Brij-96, 1% (vol/vol) Thesit, and 1% (vol/vol) CHAPS. These results are consistent with a level 1 direct interaction.⁴ 

As TSSC6 is expressed by platelets but not vascular endothelium,^1,3  we wanted to test the effect of deletion of a hematopoietic-specific tetraspanin in an in vivo tail-bleeding assay. As shown in Figure 2, there was a small but significant (P < .05; n = 20) difference in the mean tail-bleeding times for wild-type versus TSSC6 knockout mice. Consistent with this feature, the mean volume of blood lost during the tail-bleeding time was 2-fold greater for TSSC6 knockout mice compared to wild-type mice (P < .05; n = 20). In addition, the percentage of rebleeds for TSSC6 knockout mice was significantly 4-fold higher (38% versus 9%; P < .005; n = 20) compared to wild-type mice, indicating unstable hemostasis. This is not attributed to any alteration in platelet count, as TSSC6 knockout mice have comparable platelet counts as wild-type mice.¹⁴  Based upon this in vivo bleeding defect, the TSSC6 knockout mice appear to have an underlying platelet functional defect.

To determine the consequences of TSSC6 deficiency in vivo, we tested wild-type and TSSC6^–/– mice in a model of lethal pulmonary thromboembolism induced by infusion of type 1 fibrillar collagen (data not shown). All wild-type and TSSC6^–/– mice died within 240 seconds, with more than 60% reduction in circulating platelet counts within 3 minutes of challenge (data not shown). Consistent with these observations, histologic sections of lung tissue and analysis of occlusive thrombi in the lungs reveals similar numbers of occluded vessels between wild-type and TSSC6^–/– mice (data not shown).

We studied the effect of TSSC6 deficiency on thrombus formation in wild-type and TSSC6^–/– mice using a well-characterized mouse model of arterial injury. The ferric chloride (FeCl₃) oxidative injury model was used to induce formation of free radicals to disrupt the vascular endothelium in mesenteric arterioles.¹⁸  Following application of FeCl₃ to the mesentery, platelets in both wild-type and TSSC6^–/– mice rapidly commenced interacting with the injured vessel wall and within 10 to 15 minutes after the injury, vessel occlusion occurred. However, the clotted vessels in TSSC6^–/– mice were less stable over time, with microaggregates and thrombi breaking loose from the site of injury or from platelet aggregates. Typically, TSSC6^–/– mice showed a slight delay in time to stable clot formation and reduced stability of clotted vessel formed in vivo following thrombus occlusion over time (Figure 3A-C) (P < .05). In contrast, TSSC6^–/– mice showed no abnormality in the time to first thrombi more than 20 μm, number of thrombi more than 20 μm, the increase in area or diameter over 1 minute for forming thrombi more than 20 μm (P > .05; n = 25) (data not shown). Collectively, these data demonstrate that TSSC6 is required for the secondary stabilization of platelet-rich thrombi in FeCl₃-injured mesenteric arterioles.

Based upon in vivo evidence that platelet TSSC6 contributes to the stabilization of platelet thrombus formation, we wanted to investigate the possibility that TSSC6 may modulate the function of integrin α_IIbβ₃ in murine platelets. As the formation of stable platelet aggregates is dependent upon the association of activated integrin α_IIbβ₃ in complex with its soluble ligand, fibrinogen, we examined integrin α_IIbβ₃-dependent ex vivo platelet responses. This was achieved by studying kinetics of clot retraction, platelet spreading, and platelet aggregation. As shown in Figure 4A, a delay in the kinetics of clot retraction was observed in TSSC6 knockout platelets compared with wild-type platelets. Wild-type platelets started retracting at 20 minutes and were completely retracted by 1 hour, while TSSC6 knockout platelets showed only partial retraction by 60 minutes. This defect was not attributable to reduced expression of integrin α_IIbβ₃ on the surface of TSSC6 knockout platelets, as demonstrated by flow cytometry (Figure 4B). In addition, CD44 and CD9 expression were normal on TSSC6 knockout platelets. These results indicate that the hematopoietic-specific tetraspanin TSSC6 regulates integrin α_IIbβ₃ signaling in murine platelets.

To test if the “outside-in” signaling properties of integrin α_IIbβ₃ are defective in TSCC6 knockout platelets, we next examined the formation of filopodia and spreading on fibrinogen over time using differential interference contrast (DIC) microscopy. As shown in Figure 5A-B, TSSC6^–/– platelets exhibited a significant 3-fold reduction in their capacity to spread on immobilized fibrinogen over time compared to wild-type platelets (*P < .05; n = 3). Under DIC microscopy, a larger proportion of TSSC6^–/– platelets adhered to immobilized fibrinogen typically showed rounded morphology with limited filopodia and spreading (Figure 5A-B). This defect is related to integrin α_IIbβ₃-dependent platelet spreading and not platelet adhesion, as an equivalent number of platelets were observed to adhere to immobilized fibrinogen over time for both wild-type and TSSC6^–/– platelets (P > .05; n = 3) (Figure 5C). These results indicate that TSSC6^–/– platelets have restricted cytoskeletal reorganization when adhered to immobilized fibrinogen.

As integrin α_IIbβ₃ is required in platelet aggregation, we next examined the platelet aggregation responses of TSSC6 knockout platelets compared with wild-type platelets using a range of platelet agonists. As shown in Figure 6, wild-type and TSSC6 knockout platelets display equivalent platelet aggregation profiles at higher concentrations of PAR-4 (250 μM), calcium ionophore (2.5-10 μg/mL), type 1 acid-soluble collagen (10-20 μg/mL), and ADP (5-20 μM). In contrast, at lower concentrations of PAR-4 (125-150 μM) and type 1 collagen (5 μg/mL), TSSC6 knockout platelets showed reduced platelet aggregation responses and, in some cases, evidence of disaggregation in the secondary phase of platelet aggregation. This defect in TSSC6 knockout platelets also was observed with low-dose thrombin (≤ 0.25 U/mL) in a washed platelet system (data not shown). Therefore, based upon these results, TSSC6 knockout platelets display normal amplitude and slope of platelet aggregation responses at higher concentrations of PAR-4, ADP, calcium ionophore, and type 1 collagen. However, TSSC6 knockout platelets display reduced amplitude and slope of platelet aggregation responses at lower concentrations of PAR-4 and type 1 collagen.

As TSSC6^–/– platelets primarily show a defect in “outside-in” integrin α_IIbβ₃-mediated signaling, we wanted to confirm that TSSC6^–/– platelets have normal “inside-out” integrin α_IIbβ₃-mediated signaling properties. To test for a defect in integrin α_IIbβ₃ activation, the ability of wild-type and TSCC6^–/– platelets to bind FITC-fibrinogen and JON/A mAb under resting conditions and following CRP (2.5-5 μg/mL), thrombin (0.5-1 U/mL), PMA (20 μM), ADP (10-20 μM), and ADP (10-20 μM) + epinephrine (20 μM). The binding of soluble FITC-fibrinogen or JON/A mAb was assessed by flow cytometry. As shown in Figure 7A-B, agonist-induced stimulation of wild-type and TSSC6^–/– platelets demonstrated a similar ability to bind soluble FITC-fibrinogen as well as JON/A mAb, which recognizes the active conformation of murine integrin α_IIbβ₃. In addition, we wanted to examine the kinetics of alpha granule release of TSSC6 knockout platelets. As shown in Figure 7C, the surface expression of P-selectin on thrombin and PAR-4 stimulated platelets derived from wild-type and TSSC6 knockout platelets were equivalent over a range of thrombin (0.5-5 U/mL) and PAR-4 (100-400 μM) concentrations. Therefore, based upon these results, it would appear that TSSC6^–/– platelets have normal “inside-out” integrin α_IIbβ₃-mediated signaling properties and normal alpha granule release.

An emerging theme in tetraspanin research is the concept that tetraspanins associate with each other and with transmembrane receptors to form tetraspanin webs. This concept is supported by recent studies in tetraspanin knockout mice where CD37^–/–, CD151^–/–, CD81^–/–, and TSSC6^–/– mice showing similar phenotypes of hyperproliferative T cells^14,16,19,20  and CD9^–/– and CD81^–/– mice displayed similar phenotypes of sperm-egg fusion fertility defects.^21-24  In addition, the “outside-in” integrin α_IIbβ₃ signaling defect and unstable hemostasis phenotype of CD151^–/– mice¹³  is somewhat similar to the phenotype of TSSC6^–/– mice reported in this study.

TSSC6 appears to be crucial in the negative regulation of peripheral T-lymphocyte proliferation. TSSC6^–/– T cells demonstrated enhanced responses to concanavalin A, anti-CD3 alone, and anti-CD3 + anti-CD28. This heightened responsiveness by TSSC6^–/– T cells was attributed to increased interleukin 2 (IL-2) production following T-cell antigen receptor stimulation.¹⁴  However, its functional role in hematopoietic cells is undefined. A recent report suggested that TSSC6 may be expressed by platelets based upon detection of TSSC6 mRNA transcript in K562 cells differentiated into megakaryocytic lineage.^1,3  In this study, we provide evidence for the presence of TSSC6 expression both on the surface as well as within intracellular pools of platelets. Furthermore, TSSC6 was found to be constitutively associated in a physical complex with the major platelet glycoprotein, integrin α_IIbβ₃ in resting platelets (Figure 1). From a functional perspective, TSSC6^–/– mice displayed evidence of unstable hemostasis in vivo (Figure 2), and TSSC6^–/– platelets demonstrated features of an “outside-in” integrin α_IIbβ₃ signaling defect in vitro (Figures 4, 5, 6, 7). Finally, TSSC6 was found to play a role in thrombosis based upon instability in platelet thrombi formed in a murine model of FeCl₃-induced in vivo vascular injury (Figure 3A-C).

Previous studies have demonstrated that in normal resting platelets, tetraspanin superfamily members are located in different subcellular compartments. CD9 is highly expressed on the platelet surface and is located in alpha granules within the cytoplasms of platelets together with integrin α_IIbβ₃, fibrinogen, and von Willebrand factor (VWF).⁶  In contrast, CD63 is located on the membranes of dense granules and lysosomes.⁷  In both cases, platelet activation resulted in relocation of these tetraspanin superfamily members to the plasma membrane, where they form a complex with integrin α_IIbβ₃. Based upon our current study, TSSC6 surface expression on platelets was up-regulated by stimulation with thrombin, indicating release of TSSC6 from an intracellular pool. This observation that TSSC6 and integrin α_IIbβ₃ are stored in the same intracellular granular pool and migrate to the plasma membrane after platelet stimulation lends support to the concept of a close constitutive association between TSSC6 and integrin α_IIbβ₃. Whether TSSC6 interacts directly or indirectly with integrin α_IIbβ₃ is not known, but it is likely that the relationship is complicated, potentially involving other tetraspanin multimolecular complexes. The exact number of copies of TSSC6 per platelet has not been determined; however, based upon quantitative fluorescence-activated cell sorting scanner (FACS) data, it would appear to be less than integrin α_IIbβ_3, CD9, and CD151, which are expressed at a higher density (magnitude of expression: integrin α_IIbβ₃ > CD9 > CD151 > TSSC6).

The fact that tetraspanin superfamily members can form lateral associations with integrins suggests that TSSC6 plays a role as an organizer of multimolecular complexes involving integrin α_IIbβ₃ to regulate its adhesive/signaling events. Following this, our studies revealed that TSSC6^–/– mice displayed features of unstable hemostasis as shown by a tendency for rebleeds, as well as increased blood loss and tail-bleeding times in vivo. In addition, TSSC6^–/– platelets have an “outside-in” integrin α_IIbβ₃ signaling defect shown by a delay in kinetics of clot retraction and restricted cytoskeletal reorganization in platelet spreading on fibrinogen. Many of these features resemble the phenotypes observed in the tetraspanin superfamily member CD151^–/– mice¹³  and the integrin α_IIbβ₃ Y747759F mice.²⁵  In addition, recent studies using antibody cross-linking of CD63 suggests that CD63 also may be functionally linked with integrin α_IIbβ₃ in platelets.⁷ 

In the in vivo injury model, TSSC6^–/– mice exhibited secondary instability in formed platelet thrombi, resulting in frequent embolization compared with wild-type mice. As TSSC6 is restricted to hematopoietic lineages, it would suggest that platelet TSSC6 is required for maintaining stable platelet thrombus formation in vivo. It would appear that TSSC6 contributes to the formation of stable integrin α_IIbβ₃-mediated platelet aggregates within a thrombus and particularly in the anchorage of platelet aggregates to the injured vessel wall interface. In these experiments, cohorts of large platelet aggregates were observed to be detaching from the entire thrombus as well as the injured vessel wall interface under high shear flow conditions. Based upon these observations, it is most likely that TSSC6 plays a key role at high shear in platelet aggregation rather than fibrin formation that stabilizes the growing thrombus.

The embolization observed in the absence of TSSC6 may have important clinical implications since fatal pulmonary embolus formation has been reported in afibrinogenemia patients.^26,27  At this stage, no naturally occurring TSSC6-deficient patients have been reported to test the possibility that spontaneous bleeding problems are encountered due to the reduced inability to form stable platelet plugs at site(s) of injury.

Several studies have highlighted that tetraspanins do not modulate integrin-dependent static cell adhesion, but regulate integrin adhesion strengthening.²⁸  Tetraspanin CD151 was found to play a key role in regulating the adhesion strengthening of the integrin α₆β₁, particularly involving the C-terminal region of CD151.²⁹  In addition, the CD81 tetraspanin was found to facilitate the adhesion strengthening of vascular-cell adhesion molecule 1 (VCAM-1) under shear flow conditions.³⁰  Taken together, these studies suggested that tetraspanins may exert their effect by regulation of the “outside-in” properties of integrin-mediated signaling.

This study places into context the functional importance of a hematopoietic-specific tetraspanin, TSSC6, in stabilizing arterial thrombi but not thrombus growth in vivo involving a functional relationship with integrin α_IIbβ₃ “outside-in” signaling in platelets. While mechanisms of early events in platelet activation are well known, many of the late events that control platelet thrombus growth and stability are still poorly defined. One of the key unanswered questions raised by Brass and colleagues is which mechanisms prevent destabilization of the hemostatic plug and clot retraction during the time needed for wound healing to occur.³¹  Several molecules have been implicated in contact-facilitated events in platelets that regulate thrombus growth and stability, including platelet-derived CD40L and Eph kinases/ephrins, that also regulate integrin α_IIbβ₃ “outside-in” signaling events.^32,33  Specifically, CD40L^–/– mice showed a delay in platelet thrombus growth and blood vessel occlusion following vascular injury combined with a decrease in thrombus stability.³²  These previous studies suggested that CD40L behaves as an integrin α_IIbβ₃ ligand that is necessary for stability of arterial thrombi. Based upon the results of our current study, the hematopoietic-specific tetraspanin TSSC6 appears to be a new integrin α_IIbβ₃ partner that modulates its “outside-in” signaling properties necessary for stability but not growth of arterial thrombi in vivo. This TSSC6:integrin α_IIbβ₃ interaction in platelets is therefore likely to be an important mechanism for stabilization of the hemostatic plug required to stop bleeding and subsequent clot retraction that is required during the process of wound healing.

L.-M. Lau is a recipient of a National Heart Foundation Biomedical PhD Scholarship and is a postgraduate student of the University of Melbourne.