Short Polyphosphates Inhibit the Procoagulant Activities of Long-Chain Polyphosphate: A Novel Role for Platelet-Derived Pyrophosphate?.

Inorganic polyphosphates are negatively charged, linear phosphate polymers that influence hemostasis via accelerating factor V activation, triggering the contact pathway, and enhancing fibrin polymerization. The latter two effects require long-chain polyphosphates for optimal activity (>1000mers for activating the contact pathway and >250mers for enhancing fibrin polymerization). We explored whether short polyphosphates could inhibit the procoagulant effects of long polyphosphate polymers.

Polyphosphate was size-fractionated by gel electrophoresis. “Long-chain polyphosphate” (LCP) was a heterogeneous polyphosphate preparation containing polymers ranging from 400 to several thousand phosphates long. Short-chain polyphosphate preparations were narrow fractions of homogeneous size. We also tested additional phosphate-containing molecules including ADP, ATP, monophosphate, pyrophosphate (PP_i), and triphosphate. Clotting assays were performed using pooled normal plasma spiked with 0–20 μM LCP and 0–500 μM small (poly)phosphate; clotting was initiated by factor Xa plus CaCl₂, or by CaCl₂ alone (contact pathway). Fibrin was formed by clotting 2.6 mg/ml fibrinogen with 10 nM thrombin in the presence of 0–500 μM small (poly)phosphate plus 150 μM LCP and CaCl₂.

Small polyphosphates (size range 23–83mer) reduced the ability of LCP to trigger the contact pathway of blood clotting, while monophosphate, PP_i, triphosphate, ADP, and ATP all failed to influence the procoagulant activity of LCP. None of the small (poly)phosphates antagonized the ability of LCP to enhance factor V activation. Interestingly, monophosphate, PP_i and triphosphate all inhibited the ability of LCP to enhance fibrin clot structure, with PP_i being the most potent. Although ADP and ATP contain di- and tri-phosphates, they did not recapitulate the potent inhibitory activity of PP_i. On the other hand, PP_i had no measurable effect on the turbidity of fibrin clots formed in the absence of LCP.

Short-chain polyphosphates inhibit the ability of LCP to initiate the contact pathway of coagulation. Platelet dense granules contain abundant PP_i which is secreted in response to platelet agonists, although biological roles for platelet-secreted PP_i are unclear. We propose that PP_i is a novel modulator of fibrin clot structure, acting to regulate the effects of longer-chain polyphosphates on fibrin fibril formation.

Smith:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.