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W G Murphy

Publications and source records attributed to W G Murphy.

63 records · Page 4Linked to original sources
PubMed1988

Heparin-induced thrombocytopenia: laboratory studies.

This report describes studies into the pathophysiology of heparin-induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin-induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule.

Antibodies, Monoclonal↗
PubMed1987

Platelet IgG Fc receptor.

The glycoprotein localization of the platelet binding site for the Fc IgG has been the subject of debate. We attempted to resolve this issue by relating the binding of radiolabeled IgG immune complexes composed of heat-aggregated IgG to platelets from healthy individuals; an individual with Bernard-Soulier syndrome lacking glycoproteins IIb and IX; and a patient with Glanzmann's thrombasthenia lacking glycoproteins IIb and IIIa. The binding of IgG complexes to platelets was determined by measuring the specific binding of radiolabeled heat-aggregated IgG to washed platelets in a plasma-free mileu. 125I aggregated IgG bound to normal platelets in a saturable and concentration-dependent fashion. Specific binding could be inhibited by a 50-fold excess of purified Fc, but not by F(ab')2. Identical binding curves were obtained by using platelets from a patient with Glanzmann's thrombasthenia and a patient with Bernard-Soulier syndrome, indicating that the platelet Fc receptor is not carried on glycoproteins Ib, IIb, IIIa, or IX. We then measured the binding of radiolabeled detergent-solubilized platelets to IgG fixed to a solid matrix. A 40-kD platelet fragment bound to the immobilized IgG following passage across a density gradient. Confirmation of the Fc specificity of the interaction was shown by inhibition of platelet glycoprotein binding by excess IgG or purified Fc but not F(ab')2. The electrophoretic mobility decreased slightly after reduction, which indicated the existence of at least one intrachain disulfide bond. Treatment with high salt solutions or urea did not solubilize the receptor, which indicated that it was an integral protein. Enzyme studies showed that the platelet Fc receptor was not digested by neuraminidase, but neuraminidase treatment altered mobility by about 3%. In addition, treatment of platelets with trypsin or pronase did not affect its function as measured by the binding of 125I-IgG aggregates to enzyme-treated platelets, but did prevent its detection when using radioimmunoprecipitation studies. The platelet Fc receptor is a 40-kD, integral protein without interchain disulfide bonds.

Bernard-Soulier Syndrome↗
PubMed1987

Relationship between platelet aggregating factor and von Willebrand factor in thrombotic thrombocytopenic purpura.

The pathophysiology of the platelet thrombotic disorder, thrombotic thrombocytopenic purpura (TTP), is not well understood. Two apparently unrelated laboratory abnormalities have recently been described in patients with TTP: a platelet aggregating factor and abnormalities in von Willebrand factor (vWF). Although an interaction between these two abnormalities has been postulated to participate in the disease, this has not been proved. In this report we describe studies on a patient with relapsing TTP. These studies demonstrate that a consistent relationship exists between the platelet aggregating factor present in the patient's serum and vWF. The patient had chronic low-grade thrombocytopenic and schistocytic haemolytic anaemia that could be temporarily cured by infusions of plasma and certain other blood products. During acute exacerbations of the illness, a platelet aggregating factor was detectable in the patient's serum and this was associated with the loss of the larger multimers of vWF. During remissions of the illness, abnormally large multimers of vWF were present. The results of this study support the concept that a platelet aggregating factor plus large multimers of vWF participate in the acute platelet thrombi that characterize TTP.

Anemia, Hemolytic↗
PubMed1987

Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura.

Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1-arginine chloromethyl ketone. These studies suggested that the platelet-activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.

Blood Platelets↗
PubMed1987

Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura.

Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

Adenosine Diphosphate↗