Polyphosphate Enhances Fibrin Clot Structure.

Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V. Our previous studies found that polyP did not affect the time to clot formation when plasma was clotted with thrombin, however, suggesting that polyP exerts its procoagulant actions upstream of thrombin. We now report that polyP enhances fibrin clot structure.

Methods: Purified fibrinogen and polyP were preincubated for 15 min in multiwell plates in buffer containing CaCl₂, after which clotting was initiated by adding 0.1 to 8 nM thrombin and fibrin clot formation was evaluated by quantifying the change in turbidity (A₄₀₅). Mass-length ratios were calculated from scans of A₄₀₀ to A₈₀₀. The effect of polyP on fibrinolysis was examined by adding 8 nM plasmin to the reaction mixtures immediately prior to thrombin. Scanning electron microscopy (SEM) was employed to visualize clot structure, and time courses of covalent fibrin cross-linking were assessed by SDS-PAGE.

Results: PolyP had no effect on time to clot formation, but clots formed in the presence of polyP had markedly (up to threefold) higher turbidity than clots formed in the absence of polyP (see figure), irrespective of thrombin concentration. The increased turbidity in the presence of polyP was calcium-dependent and was enhanced when fibrinogen, CaCl₂, and polyP were preincubated for up to 15 min prior to initiation of clotting with thrombin. PolyP increased the mass-length ratio of fibrin, and SEM confirmed that fibers formed with polyP were thicker than those formed without polyP. The ability of polyP to enhance fibrin clot turbidity was independent of factor XIIIa activity, and polyP did not alter the rate or extent of covalent fibrin cross-linking by factor XIIIa. When plasmin was included in clotting reactions containing polyP, mean times to 50% clot lysis were 28.5 ± 0.8 min for clots without polyP but 120.4 ± 5.6 min for clots with polyP.

Conclusions: PolyP alters polymerization of fibrin, resulting in fibers of higher mass-length ratio that are lysed more slowly. This effect is calcium-dependent and is enhanced by preincubation of fibrinogen with calcium and polyP. Release of polyP from activated platelets or infectious microorganisms may therefore enhance fibrin clot structure.

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