High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa.

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.

Separation of different receptor-mediated effects of a prostaglandin H2 analogue (U46619) on human platelets by means of human granulocytic elastase and chymotrypsin.

Previous investigations indicated two classes of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors on human platelets and suggested that shape change and myosin light chain phosphorylation correlated with the occupancy of high affinity receptors while serotonin release was related to a putative low affinity binding component (Morinelli TA et al., Am J Physiol 253: H1035-H1043, 1987). The current study shows that chymotrypsin destroyed three receptor-mediated responses of platelets to U46619 (a TXA2/PGH2 agonist), i.e. shape change, myosin light chain phosphorylation and serotonin release. Human granulocyte elastase selectively inactivated platelet ability to release serotonin following stimulation with U46619, but it did not affect significantly shape change and myosin light chain phosphorylation. In conclusion, it is possible to separate different receptor-mediated effects of U46619 on human platelets by means of human granulocytic elastase and chymotrypsin.

Loss of platelet fibrinogen receptors during clinical cardiopulmonary bypass.

In 10 patients, cardiopulmonary bypass decreased the number of fibrinogen binding sites from 31,730 +/- 12,802 per platelet to 18,590 +/- 9,644 per platelet. Bypass also decreased the amount of the platelet membrane glycoprotein IIIa, which is part of the fibrinogen receptor complex, from 17.1 +/- 3.6 ng/10(9) platelets to 12.9 +/- 4.7. The fibrinogen binding constant did not change. Platelet sensitivity to adenosine diphosphate did not change; however, template bleeding times increased from 5.2 +/- 1.5 minutes before bypass to 8.5 +/- 2.3 minutes after bypass. Analysis of detergent washings from the perfusion circuit after bypass in five patients indicated that platelet material remains attached to the surface as membrane fragments and degranulated platelets. These data further elucidate the mechanism of platelet loss and dysfunction during cardiopulmonary bypass and highlight the importance of platelet membrane fibrinogen receptors and surface adsorbed fibrinogen in this process.

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.

Biological and biochemical characteristics of serum fraction effluent from xenografts and inhibiting leukocyte migration.

Serum effluent from the hyperacutely rejected rabbit-to-dog liver graft strongly inhibits migration of peripheral blood leukocytes. Fractionation of this serum on DEAE-Sephadex A-50 revealed the most active fraction to be that containing IgG. This finding supports our view that immune complexes formed during the hyperacute rejection of xenografts are mainly responsible for the inhibition of migration of peripheral blood leukocytes.

Immune complexes in children with different forms of glomerulonephritis.

The occurrence of circulating immune complexes was tested in 43 children with different forms of glomerulonephritis (GN). The presence of immune complexes was demonstrated in sera of 6 out of 10 patients with GN of hepatitis B etiology, in 6 out of 11 patients with GN of bacterial etiology, and in 3 out of 10 patients with GN of unknown etiology. Immune complexes were not found in sera of 3 patients with Schönlein-Henoch disease, in 6 patients with lipoid nephrosis, and in 3 patients with GN and hepatitis B as a coexisting infection. In a patient with membrane-capillary GN which appeared after infection with hepatitis B virus we demonstrated immune complexes of M.W. 4.0 X 10(6) and 7.5 X 10(5).

[Molecular markers of hemostasis activation in children with nephrotic syndrome]. / Molekularne markery aktywacji hemostazy u dzieci z zespolem nerczycowym.

Vein and arterial thrombosis is a rather rare but potentially life-threatening complication of nephrotic syndrome (n.s.). None of the specific markers of hypercoagulability state in n.s. have been identified. The aim of the study was to estimate plasma parameters of prothrombic state in children with n.s. Ten children aged from 3 to 10 yrs (mean 5.7 +/- 2.5) with recurrence of n.s. and 10 healthy controls matched for age and sex were studied. In all children with n.s. prothrombin fragments F 1+2, D-dimers (D-d), thrombin-antithrombin III complexes (TAT) and whole blood clotting time thrombin time, prothrombin time, plasma fibrinogen and platelet count were determined in the hypovolemic state (before therapy was started), in the normovolemic state (after plasma expander was used) and in the course of anticoagulation treatment (two week dicumarol therapy). In comparison with healthy controls all children with recurrence of n.s. in the hypovolemic state showed a significant (p < 0.05) increase of D-d (910 vs 500 ng/ml), TAT (14.26 vs 2.6 ng/ml), F 1+2 (5.68 vs 0.79 nmol/l), plasma fibrinogen (592 vs 272 mg/dl), platelet count (632 vs 275 x 10(9)/l) and shortening of WBCT (30.0 vs 35 s). Plasma volume expansion produced by dekstran was followed by a moderate decrease of all parameters. Two-week anticoagulant treatment had no impact on estimated haemostasis parameters.