A platelet-aggregating activity was found in many snake venoms, predominantly those of the genus Bothrops, that is apparent only in the presence of the platelet-aggregating von Willebrand factor of plasma. It is designated "venom coagglutinin." The coagglutinin can be largely separated from the thrombin-like enzyme of the venoms by ionexchange chromatography. The venom factor acts on formaldehyde-fixed platelets and is effective with decalcified, heparinized, and afibrinogenemic plasmas but not with severe von Willebrand disease plasmas or with normal plasmas in which the von Willebrand factor has been neutralized by specific antibodies. Use of this coagglutinin permits the assay of von Willebrand factor without the many disadvantages of the ristocetin test. The coagglutinin is active with human, dog, pig, and bovine plasmas and with platelets of any one of these species. This broad-spectrum activity without regard to species contrasts with the ristocetin-resistance of many combinations of plasma and platelets from various species. The assay provides a procedure for studying human, porcine, and canine von Willebrand disease. The lack of species specificity of the coagglutinin suggests that it may be a universal activator of the von Willebrand factor-platelet reaction.

A one-stage macroscopic test for platelet agglutination was used to study the effect of thrombin and thrombin-cation mixtures on washed platelets. Conclusions regarding platelet agglutination are as follows: (a) Canine, bovine, or human thrombin alone does not cause agglutination of canine or human platelets. (b) Thrombin with calcium or magnesium causes rapid platelet agglutination. Both calcium and magnesium are active at physiologic concentrations. Divalent manganese or cadmium ions can be substituted for calcium or magnesium. (c) The agglutination reaction is affected but little by the species of origin of thrombin or platelets, or by variations in ionic strength or pH over a broad range. (d) Temperature at which the reaction is carried out is critical; optimal temperature for the test is 28°C. (e) Agglutination is inhibited by high ionic strength, by pH values outside the range 6.4–8.6, and by temperatures outside the range 25–28°C. High concentrations of calcium have a specific inhibitory effect. (f) Platelet agglutination time is as sensitive an index of thrombin concentration as is the fibrinogen clotting time. A comparison is made between divalent cations which influence platelet agglutination induced by thrombin, TAg', and TAg. A similar comparison is made of cations influencing the action of thrombin on the "substrates," fibrinogen, TAMe, and platelets.

An in vilro test cell is described which permits exposure of artificial surface5 in sheet or film form to native human blood in the absence of a blood-air interface. Evaluation of Cuprophane, polyethylene, Silastic, and silicone-coated glass in the in vitro cell showed Cuprophane and silicone-coated glass to produce the least activation of the intrinsic coagulation system, while Silastic produced the greatest degree of activation. Polyethylene gave results intermediate between those obtained with the other materials. Each of these four different materials wm evaluated in the elliptical cell test system multiple times with blood from 13 different donors.Our data suggest that with the present test system and the proper experimental design, one might expect that, "on the average," performance of 36 tests on each of two materials will be sufficient for a 10% difference in their compatibility with blood to be statistically significant at the 5% level. The number of tests is 26 for significance at the 10% level.

SummaryThrombosis of the microvasculature has been recognized as the end product of organ rejection, but the exact biological pathway through which this occurs has not been clarified. Normal, factor VII deficient and heterozygous hemophilic (factor VIII) dogs were grouped to study the intrinsic and extrinsic clotting and platelet mechanisms during unmodified renal allograft rejection. The observed alterations of the hemostatic mechanisms are related to the changes observed in the microvasculature. Six groups of donor-recipient animals were studied: Group I - autografts (control); Group II - normal to normal allografts with bilateral nephrectomy; Group III - heterozygotes for factor VIII deficiency; Group IV - normal to normal allografts with unilateral nephrectomy; Group V - normal to factor VII deficiency without nephrectomy; and Group VI - normal to normal allografts with unilateral nephrectomy and dipyridamole. Each engrafted animal was followed pre- and posttransplantation for change in the blood clotting factors, fibrin split products, platelets, white blood cells, renal function and microvasculature. The animals with factor VII deficiency rejected in a similar fashion as the control animals. The group with impaired factor VIII synthesis and platelet function had longer survival times. These data suggest that the intrinsic clotting pathway and platelets are the primary mechanism through which thrombosis occurs secondary to immune injury.