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Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445–733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445–733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.

A specially designed rotational viscometer was used to investigate the effects of the antiplatelet agent PGI2 in combination with theophylline on the response of human platelets subjected to shear stress. Samples of citrated platelet-rich plasma (PRP) were exposed to shear stress in the viscometer for a period of 5 min at 23 degrees C. The levels of stress studied ranged from 50 to 300 dynes/sq cm. Pretreatment of the platelets with 0.01 microM PGI2 and 500 microM theophylline before exposure to shear stress caused a large reduction in shear-induced platelet aggregation. However, it was also observed that the PGI2-- theophylline pretreatment concomitantly caused a large increase in shear-induced platelet lysis and serotonin release at stress levels equal to or greater than 150 dynes/sq cm. This observed increase in platelet fragility may have important implications for clinical applications of PGI2. The results are discussed and compared to those obtained in prior work in which platelets were pretreated with acetylsalicylic acid or with PGE1.

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.

von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF. There was no significant relationship between either gestational age at time of delivery or birth weight and likelihood of finding ULvWF multimers in cord PPP samples. No maternal PPP sample contained ULvWF multimers. Serial heelstick samples from 16 preterm and term neonates were analyzed for 8 weeks. ULvWF multimers disappeared from the PPP of ten of the neonates during this time. The PPP of four neonates had vWF patterns similar to those in normal adult PPP throughout the sampling period. The ULvWF multimeric forms of fetal and neonatal PPP samples were similar to those constitutively released from endothelial cells. They were not as slowly migrating in a very porous 0.5% agarose gel system as the ULvWF multimers released from Weibel-Palade bodies in response to the calcium ionophore A23187. A vWF protomer was present in 97% of fetal samples, 83% of cord blood specimens, and 11% of neonatal heelstick samples, but was not found in any maternal sample. These results indicate that control mechanisms operative in older children and adults to prevent circulation of ULvWF multimers and vWF protomeric forms are normally acquired late in uterine life or during the neonatal period. ULvWF multimers, which are normal components of fetal, most cord, and some neonatal plasma samples, may contribute to in utero and postnatal hemostasis.

A 72-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.

The preparation and properties of a Mg2+-dependent, (Na+ + K+)-stimulated ATPase from platelets are described. The enzym system require Mg2+ for activity, is stimulated maximally by the presence of Na+ and K+ in a ratio of 11.5/1, has a pH optimum 7.30–7.40, and is half-maximally inhibited by ouabain at a concentration of 0.15 μM. The Km for ATP is approx. 0.4 mM. Enzyme activity is localized in both the “membrane” and “membrane-granule” subcellular fractions. Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity is inhibited by ADP (which induces platelet aggregation) and by Pi. The inhibition by ADP is apparently not competitive with respect to ATP. Mg2+-dependent, K+-stimulated p- nitrophenyl phosphatase activity (a model for the externally-oriented K+-phosphatase portion of Mg2+-dependent, (Na+ + K+)-stimulated ATPase) is also inhibited by ADP. GDP, which does not induce aggregation of platelets, has no effect on the Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity. It is postulated that ADP interacts with the external platelet membrane surface to inhibit Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity with a resultant decrease in cation transport and membrane potential. This phenomenon may play a role in platelet aggregation.