Antithrombin III. II. Comparison of different substrates and thrombin preparations

Antithrombin III (AT-III) activity may be measured by amidolytic methods; the synthetic chromogenic substrates, Chromozym® TH, S 2238 and S 2160, which are available at present for the measurement of thrombin activity, are compared in the assay of AT-III activity using an automated method. In addition, AT-III activities from patient plasmas are also compared using bovine and human thrombin. After suitable modification of the assay system no significant differences were found using all three substrates when either human or bovine thrombin was used. Commercially available lyophilized control plasmas were tested for possible use as a standard plasma. These plasmas were not preferred as all showed a decreased level of AT-III activity compared to that of fresh frozen pool plasma     

Antibodies against Platelet Membrane Glycoproteins II. INFLUENCE ON ADP- AND COLLAGEN-INDUCED PLATELET AGGREGATION, CROSSED IMMUNOELECTROPHORESIS STUDIES AND RELEVANCE TO GLANZMANN''S THROMBASTHENIA

In Glanzmann's thrombasthenia glycoproteins IIb and IIIa are missing or strongly reduced and aggregation to ADP, collagen and thrombin is impaired. Antibodies against glycoproteins IIb and IIIa did not entirely induce a thrombasthenia-like state in normal platelets. However, they did strongly inhibit collagen-induced aggregation and inhibited the second wave of aggregation induced by ADP. Crossed immunoelectrophoresis studies using Triton X-100 extracts of whole platelets with these antibodies gave a single immunoprecipitate. This immunoprecipitate was absent when similar studies were carried out with thrombasthenic platelets. Platelet antibodies gave a number of immunoprecipitates with normal platelets and differences were observed with thrombasthenic platelets, the most notable of which was a marked reduction in one of the major immunoprecipitates. These results provide further evidence that glycoproteins IIb and IIIa are involved in the latter stages of platelet aggregation.     

Antibodies against Platelet Membrane Glycoproteins

S ummary. Platelet membrane glycoproteins have been isolated by lectin-affinity chromatography and antibodies prepared against them. Platelets that have lost glycocalicin no longer respond to ristocetin-human VIIIR:WF, bovine VIIIR: WF, or to glycocalicin or glycoproteins la and lb antibodies but are still agglutinated by glycoproteins IIb and IIIa antibodies. Glycoproteins la and Ib and glycocalicin antibodies, IgG and Fab fragments, inhibited ristocetin-human VIIIR: WF-induced aggregation of fixed, washed platelets and of platelets in plasma while glycoproteins IIb and IIIa antibodies were without effect.
Cross immunoelectrophoretic studies showed that glycocalicin was present on whole platelets in only trace amounts. Glycocalicin antibodies, however, recognized a slower migrating component. Platelets incubated in an EDTA-free medium no longer respond to ristocetin-human VIIIR: WF. Membranes isolated from such platelets contained glycocalicin which cross-reacted with a remnant of the slower migrating component. Glycoproteins la and Ib antibodies gave more complex patterns but it was possible to identify the slower moving component recognized by the glycocalicin antibodies.
These results show that glycocalicin is not normally found as such on whole platelets but is present as a precursor which is most likely glycoprotein Ib. On degradation of this precursor, glycocalicin is released from the membrane and VIIIR: WF-receptor activity is lost.     

Antibodies against platelet membrane glycoproteins

Glycocalicin has been proposed as the common platelet receptor for both ristocetin-human VIIIR:WF and thrombin-induced platelet aggregation. Platelets which have lost glycocalicin do not respond to either ristocetin-human VIIIR:WF or bovine VIIIR:WF. Using antibodies to the platelet membrane glycoproteins Ia and Ib, IIb and IIIa, and glycocalicin we show that the Fab' fragments of anti-glycoproteins Ia and Ib and anti-glycocalicin IgG totally inhibited bovine VIIIR:WF-induced platelet aggregation, while those from anti-glycoproteins IIb and IIIa IgG were without effect. Thrombin-induced platelet aggregation was strongly inhibited by the Fab' fragments of anti-glycoproteins Ia and Ib IgG and anti-glycocalicin IgG, indicating that these glycoproteins play a major role in thrombin-platelet interaction. Fab' fragments of anti-glycoproteins IIb and IIIa IgG inhibited thrombin-induced platelet aggregation to a lesser extent implying that these glycoproteins are also somehow involved in the platelet response to thrombin perhaps as fibrinogen receptors. The data presented here give further support to the proposal that ristocetin-human VIIIR:WF and bovine VIIIR:WF share a common receptor on the platelet surface and indicate that this structure plays an important role in thrombin-induced platelet responses.