Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.
Summary1. With the aid of isotopie technique, it was shown, that fibrinogen, fibrinogen-fibrin intermediates and fibrinogen degradation products (FDP) may form soluble complexes, which are unclottable by thrombin.2. These complexes may be precipitated by protamine sulphate or by cooling to 4° C (par a coagulation),3. The above findings are discussed in regard to their significance in the pathology of the acute defibrination syndrome :a) Paracoagulation by protamine sulphate without addition of thrombin occurs when defibrination is caused by excessive intravascular clotting, while in cases of the syndrome due to proteolytic digestion of fibrinogen, paracoagulation occurs after combined action of thrombin and protamin sulphate.b) Formation of the complexes described above may explain the occurence of cryofibrinogens in various pathological states.c) Formation of these complexes is postulated to be an important factor involved in the development of the hemostatic defect. It may be as well the expression of a defence mechanism against intravascular fibrin formation by rendering unclottable the intermediates of the fibrinogen-fibrin conversion.
The presence of human fibrinogen in suspensions of washed human platelets is a requirement for ADP-induced platelet aggregation. Digestion of fibrinogen with plasmin destroys this function of the protein. The high solubility fraction of Kabi fibrinogen, fragment X (stage 1) and framgent X (stage 2), are two, eight, and ten times, respectively, less potent in promoting ADP-induced platelet aggregation, as compared with intact fibrinogen. Fragments Y and D and the mixture of reduced and carboxymethylated chains of human fibrinogen do not support ADP-induced platelet aggregation at all. SDS polyacrylamide gel electrophoresis of nonreduced and reduced fibrinogen and its derivatives indicates that the intact fibrinogen molecule is essential for ADP-induced platelet aggregation. It is suggested that the carboxy-terminal part of the Aalpha chain and possibly also the amino-terminal part of the Bbeta chain are required for the platelet aggregation-promoting function of fibrinogen.
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