Preferential Stimulation of Contact Pathway Factors By Red Cell-Derived Microparticles: New Perspective on Hemostasis

BACKGROUND / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly.

METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP.

RESULTS.

(1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p<0.01 to p<0.05, at depletions as low as 5% normal. For example, FVIII at 15% of normal level responded to RMP by 50% reduction in R time. In contrast, RMP had little or no augmenting effect on plasmas depleted of F’s II, V, X. This suggests that RMP promote the contact pathway preferentially.

(2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation.

(3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p<0.05). This demonstrates that RMP can potentiate thrombin generation via the TF pathway, although not as robustly as by contact pathway.

CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration.