Binding of fibrinogen to human monocytes.

The interaction of fibrinogen with monocytes was studied. After stimulation with ADP (10 microM) or thrombin (1 U/ml), platelet-free suspensions of human monocytes bind 125I-fibrinogen with two different affinities in a specific and Ca2+-dependent reaction with saturation at 5.80-7.35 X 10(-7) M of added protein. The binding of fibrinogen to specific receptors on monocytes induces the procoagulant activity of these cells. Thrombasthenic cells or normal monocytes preincubated with a monoclonal antibody to the platelet glycoprotein IIb/IIIa complex (10E5) do not bind fibrinogen and have no procoagulant activity. Metabolic studies with [35S]methionine revealed that cultured monocytes actually synthesize a surface antigen precipitated by 10E5 antibody as a major band with 92,000 relative molecular weight. Our data indicate that monocytes express receptors for fibrinogen only in part related to the platelet glycoprotein IIb/IIIa complex. Furthermore, the binding of fibrinogen to monocytes enhances the cooperation of these cells in hemostasis.

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.

Interaction of platelet factor 4 with human platelets.

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.

Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells.

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.

Ticlopidine selectively inhibits human platelet responses to adenosine diphosphate.

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

Fibrinogen interaction with platelets of diabetic subjects.

Fibrinogen-platelet interaction was studied in suspensions of platelets obtained from patients with uncontrolled diabetes mellitus of long duration and from control individuals. Fibrinogen binding sites were exposed by stimulating platelets with ADP or with chymotrypsin. There was no significant difference in fibrinogen mediated aggregation between ADP-stimulated platelets of 80 control and 47 diabetic subjects. The Km values for fibrinogen mediated aggregation of ADP-stimulated platelets obtained from control and diabetic donors were 1.39 +/- 0.13 X 10(-7)M and 1.44 +/- 0.13 X 10(-7)M; the Vmax values (expressed in arbitrary light transmission units) were 87.8 +/- 3.14 and 92.8 +/- 4.5 (mean +/- S.E.M.). The analysis of variance showed no significant relationship between Km, Vmax, age and sex in control group; in patient group there was no significant relationship between Km, Vmax, age, sex, type of diabetes, presence of vascular complications and type of treatment (insulin and/or oral hypoglycemic agents). Fibrinogen mediated aggregation of chymotrypsin-treated platelets showed similar pattern in 25 control and in 25 diabetic donors. In 24 normal individuals and in 24 diabetic patients Scatchard analysis revealed 48,820 +/- 5350 fibrinogen binding sites per one normal platelet (Kd = 4.7 X 10(-7)M) and 45,350 +/- 4663 sites per one diabetic platelet (Kd = 3.5 X 10(-7)M). Our data suggest a normal pattern of interaction between fibrinogen and fully activated platelets of diabetic subjects.

Pseudopathologies of hemostasis and dental surgery.

Values of hemostasis tests are grossly abnormal in persons with factor XII deficiency, dysfibrinogenemia, the lupuslike anticoagulant, and pseudothrombocytopenia, yet these persons do not usually bleed spontaneously or even after surgery. We reviewed the records of 66 patients with laboratory diagnosis of one of the aforementioned abnormalities to determine whether they bled excessively after challenges to hemostasis such as dental extractions. Only three of them bled excessively, but two of these had other concomitant causes that could explain bleeding. Hence the dentist should not give up dental extractions or other surgical procedures when results of hemostasis tests are grossly abnormal, because these are not necessarily associated with abnormal hemostasis. Obviously, the correct interpretation of abnormal hemostasis values warrants close collaboration and reciprocal consultation between the dentist and the hematologist.

von Willebrand factor contaminating porcine factor VIII concentrate (Hyate:C) causes platelet aggregation.

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.

Exposure of fibrinogen receptors on fresh and stored platelets by ADP and epinephrine as single agents and as a pair.

Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10-15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.