Size Matters: Differential Effects of RNA and Polyphosphate on Blood Clotting

Abstract Introduction: Recently, both inorganic polyphosphate (Smith et al., PNAS 103:903-8, 2006) and RNA (Kannemeier et al., PNAS 104:6388–93, 2007) have been proposed as (patho)physiologic activators of the contact pathway in blood clotting. We also recently showed that polyphosphate of the size secreted by human platelets (approximately 75-mer) acts at two other points in the blood clotting cascade: it accelerates factor V activation and it enhances fibrin clot structure (Smith & Morrissey, Blood, in press). We now compare the ability of RNA and polyphosphate of varying chain lengths to modulate the blood clotting cascade at these three critical points: initiation, factor V activation, and fibrin polymerization. Methods: Polyphosphate was size-fractionated and its procoagulant activities were compared to those of polyinosinic acid, a synthetic singlestranded RNA (ssRNA); polyinosinic acid:polycytidylic acid, a synthetic double-stranded RNA (dsRNA); yeast tRNA or kaolin. Clotting assays were performed using purified fibrinogen, pooled normal plasma, or factor V-deficient plasma to which factor Va was added. Clotting was initiated by CaCl2 (contact pathway), factor Xa, or thrombin. Results: Long-chain polyphosphate (100-mer to 800-mer) triggered the contact pathway with a potency similar to kaolin and was about 30-fold more potent than ssRNA and some 3000- fold more potent than dsRNA or tRNA. Medium-chain polyphosphate (20-mer to 100-mer) and ssRNA both shortened factor Xa clotting times with similar potency, but dsRNA and yeast tRNA were without effect. Replacing plasma factor V with Va blocked the ability of either polyphosphate or ssRNA to shorten factor Xa clotting times, suggesting that both polymers accelerate factor V activation. And finally, when purified fibrinogen was clotted with thrombin, adding either ssRNA or polyphosphate yielded fibrin clots that were about threefold more turbid, indicating that both polymers enhance fibrin clot formation, while dsRNA and yeast tRNA were without effect. (We previously documented that polyphosphate enhances fibrin turbidity by dramatically increasing fibril diameter.) Interestingly, ssRNA significantly shortened the thrombin clotting time of purified fibrinogen, while polyphosphate had no effect on thrombin clotting times. Furthermore, the ability of polyphosphate to enhance fibrin clot structure was calcium-dependent, while ssRNA enhancement of fibrin clotting by thrombin was metal ion-independent. Conclusions: This study shows that RNA modulates critical downstream clotting functions in addition to its previously identified role in triggering the contact pathway. Long-chain polyphosphate (i.e., the size that accumulate in microorganisms, but not the size secreted by platelets) is substantially more potent than RNA in triggering the contact pathway of blood clotting. We therefore propose that polyphosphate may play an important role in host responses to pathogens by triggering the contact pathway. Polyphosphate of the size secreted by platelets had similar potency to ssRNA in accelerating factor V activation. And finally, polyphosphate of the size secreted by platelets had similar potency to RNA in enhancing fibrin clot structure, although the metal ion-dependencies of the two differed, as did their effects on thrombin clotting time. In general, ssRNA was far more potent than dsRNA or tRNA in modulating the blood clotting system.