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.

The mechanism of association and the organization of human fibrin were studied by using affinity chromatography. Insolubilized fibrinogen, fibrin monomer, and crosslinked fibrin were used to localize the binding sites on fibrinogen and fibrin derivatives. Four different polymerization sites have been distinguished. A binding site ("a"), available without thrombin action, is present on the fibrinogen fragment D domain. The complementary ("A") is inoperative in fibrinogen and requires thrombin for activation; it is located on the fibrinogen NH2-terminal domain. A third polymerization site ("b") appears to be formed by the alignment of the fragment D domains on two fibrin monomer molecules upon polymerization; this site functions without thrombin mediation and the alignment is stabilized by the Factor XIIIa-catalyzed crosslink bonds. The "b" site is complementary to another thrombinactivated site ("B") on the fibrinogen NHllterminal domain. The two thrombin activable sites, "A" and "B", are distinguishable, although they are located in the same fibrinogen domain. Activation of fibrinogen by thrombin causes the release of fibrinopeptides A and B, followed by the formation of an ordered fibrin polymer, probably due to the association of complementary binding sites (1 The purpose of the present work was to investigate the interactions of fibrinogen, fibrin, and their derivatives with short fibrin oligomers. The steric alignment of the fibrin monomer molecules appears to be an important event in the generation of a new binding site. The aligned fibrin monomers were covalently stabilized by Factor XIIIa-induced crosslinking. The prepared insolubilized crosslinked fibrin oligomers were utilized in conjunction with insolubilized fibrinogen and fibrin monomer to examine the availability of binding sites on fibrinogen and fibrin derivatives, and were used to localize and characterize fibrin polymerization sites.MATERIALS

The formation of the (DD)E complex and fragments DD and E upon proteolysis of human cross-linked fibrin was studied by timed digestions using varying amounts of plasmin. The (DD)E complex was the primary soluble degradation product released form cross-linked fibrin. This complex contained fragments DD and E1. Upon further digestion (DD)E1 complex was cleaved to (DD)E2 complex whereby only the fragment E moiety was affected. However, when fragment E2 was digested to fragment E3, dissociation of the complex occurred. Thus, fragments DD and E3 are the terminal plasmic digestion products of cross-linked fibrin. This pattern was consistent regardless of the plasmin to fibrin ratio; however, the rate of production of the terminal degradation products was directly dependent upon enzyme concentration. Digestion conditions were modified so that either the (DD)E complex or fragment DD was the predominant degradation product, allowing for the purification of these species by one-step gel filtration. The molar ratio of fragment DD to fragment E in the (DD)E complex was investigated by dissociation of the complex and by reassociation of the purified components. The (DD)E complex contains one molecule of fragment DD and one molecule of fragment E.

Fragments E1, E2, and E3 are plasmic derivatives of fibrin encompassing the NH2-terminal region of the molecule. The first two species, but not the third, can bind to fragment DD, forming a (DD)E complex, and therefore probably contain binding sites involved in the polymerization of fibrin. For localization of these sites the structure of the fragments was determined by establishing the NH2- and COOH-terminal boundaries of the molecules and using the published amino acid sequence of fibrinogen. Fragment E1 encompasses Gly-alpha 17 to Lys-alpha 78, Gly-beta 15 to Lys-beta 122, and Tyr-gamma 1 to Lys-gamma 62, this representing the intact NH2-terminal region of fibrin. Fragment E2 is an asymmetric molecule which is lacking the sequence of Gly-beta 15 to Lys-beta 53 in one beta-chain remnant. This fragment E2 also lost Lys-beta 122 from the COOH terminal of the beta chain as compared with fragment E1. These cleavages did not affect the ability of fragment E2 to bind to fragment DD. Fragment E3 was heterogeneous, the main species encompassing Val-alpha 20 to Lys-alpha 78, Lys-beta 54 to Leu-beta 120, and Tyr-gamma 1 to Lys-gamma 53. Thus, the loss of the binding function involved in the formation of fibrin clot was associated with the removal of small fragments from all three polypeptide chains: alpha 17-19 (Gly-Pro-Arg), beta 15-53 from the remaining half of the molecule, beta 121 (Leu), and gamma 54-58 (Thr-Ser-Glu-Val-Lys).

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B, and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur through the interaction of complementary binding sites on the NH2- and COOH- terminal regions of the molecules since Fragment D1, encompassing the COOH-terminals of the β and γ chains, binds to thrombin-treated NDSK which contains N2-terminals of the α , β and γ chains. A peptide of 4,200 molecular weight has been isolated from the y chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2- terminal region of fibrin monomer, thus inhibiting fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time of the reaction. The D1 peptide does not interact with NDSK, fibrinogen or Fragment D1but it binds to thrombin-treated NDSK with a Kd of 1.45 x 106 m and approximately two binding sites per molecule of NDSK have been found. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2- terminal amino acid sequence (Thr-Arg-Trp) and the COOHterminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the γ 373-410 region of the fibrinogen molecule contains a polymerization site which is complementary to the thrombin-activated site on the NH2terminal region of fibrinogen. The binding site on the Dl peptide has the characteristics of the polymerization site which is exposed and available on the COOH-terminal region of the fibrinogen molecule without any participation of thrombin.