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Biomedical subjects

A Greinacher

Publications and source records attributed to A Greinacher.

At least 91 records · Page 5Linked to original sources
PubMed1995

Characterization of the structural requirements for a carbohydrate based anticoagulant with a reduced risk of inducing the immunological type of heparin-associated thrombocytopenia.

HAT is the most frequent drug induced immune-thrombocytopenia. We recently identified multimolecular PF4/heparin complexes as the major antigen. In order to evaluate the structural requirements for formation of the antigenic complex, we chemically synthesized 13 glucan sulfates and used 5 heparin fractions (2.4-4.8 kD) and a synthesized pentasaccharide, representing the antithrombin III binding sequence of heparin, for further characterize the HAT antigen. In the presence of glucan sulfates and heparin, HAT antibodies caused platelet activation typically at low but not at high concentrations, as measured by 14C-5HT release. The concentration range giving the activation pattern depended on the degree of sulfation (DS) and molecular weight (MW) of the glucan sulfates but not on the type of glycosidic linkage of a polysaccharide. With linear glucan sulfates with a chain length of 35 monosaccharides, the critical DS to form the HAT antigen ranged between 0.60 and 1.20. Glycosidic branched glucan sulfates were able to form the HAT antigen at a lower DS and a lower MW than linear glucan sulfates. Platelet activation by HAT-antibodies in the presence of linear curdlan sulfate fractions was dependent on their MW. At a low concentration (0.01 microM) medium-size fractions (60 kD) caused platelet activation but neither small (12 kD) nor large fractions ( > 150 kD) did. At higher concentrations (2 microM) the opposite reaction pattern was observed. In the case of heparin, the optimal chain length for forming the HAT antigen is a hexadecasaccharide (4.8 kD). Antigen generation decreased with larger and smaller fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticoagulants↗
PubMed1994

Platelet response to vascular surgery--a preliminary study on the effect of aspirin and heparin.

Patients with peripheral arterial disease (PAD) demonstrate high cardiovascular mortality, which is further increased after arterial reconstruction. Enhanced platelet reactivity has been postulated for these patients. The effect of surgery and of periprocedural aspirin and heparin therapy on platelet reactivity was assessed with the Stagnation Point Flow Adhesio-Aggregometer (SPAA). The platelet adhesivity and aggregability of 44 PAD patients was quantitated perioperatively. Aspirin was administered during the entire course, low molecular weight heparin (LMWH) preoperatively and as of the fourth postoperative (pOP) day and unfractionated heparin (UH) upon surgery and three days thereafter. A group of 15 aspirin-free general surgical patients receiving LMWH and with no evidence of PAD served as controls. Plasma fibrinogen levels and platelet count were determined. The heparin-induced platelet activation (HIPA) assay for detection of heparin-associated thrombocytopenia (HAT) antibodies was also performed. Baseline values of SPAA-measured platelet reactivity (p < 0.001) and plasma fibrinogen (p < 0.01) were higher for patients as compared to controls and increased markedly after surgery. In the PAD group maximum platelet activation and fibrinogen levels coincided with a marked drop in platelet count and were concomitant to administration of unfractionated heparin. Thereby, a drop in platelet count of > 30% was observed in 25 patients (57%). The HIPA test verified HAT antibodies in 3 (12%) of these patients, two of which suffered postoperative thrombosis. In the control group significant pOP increases were noted only for plasma fibrinogen. Changes in platelet count and reactivity were minimal and nonsignificant. No thrombosis occurred and no HAT antibodies were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay.

BACKGROUND: As clinical diagnosis of heparin-associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable. STUDY DESIGN AND METHODS: The sensitivity of the heparin-induced platelet activation (HIPA) test and the platelet aggregation test (PAT) was prospectively compared by using the sera of 209 patients with the putative diagnosis of HAT. Both assays were performed concomitantly with platelets of the same four donors using a different combination of donors from day to day. Further, all sera were assessed with a platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). RESULTS: Positive results were obtained with 33 percent of sera in the PF4/heparin ELISA, with 33.5 percent of sera in the HIPA test, and with 11.5 percent of sera in the PAT. The PF4/heparin ELISA and the HIPA test showed no difference in sensitivity (p = 0.27 by McNemar's test) and were more sensitive than PAT (p < 10(-8) by McNemar's test). However, they recognized different patient cohorts. Nine HIPA-indeterminate and 12 HIPA-negative sera were positive in the PF4/heparin ELISA. Eight of the nine indeterminate sera caused platelet activation with high heparin concentrations in the HIPA test. Eleven of the 12 negative sera contained no IgG, but 9 contained IgM and 2 contained IgA HAT antibodies. Four sera that were indeterminate in the PF4/heparin ELISA and 18 sera that were negative were positive in the HIPA test. None of the sera that were positive in the PAT was missed in the HIPA test, but two of those were negative in the PF4/heparin ELISA. All sera were assessed with four low-molecular-weight heparins and a low-molecular-weight heparinoid in the HIPA test with platelets from the same four donors. Low-molecular-weight heparin caused platelet activation with positive sera in 98 percent of tests, and the heparinoid did so in 10 percent; in a further 12.8 percent, crossreactivity to the low-molecular-weight heparinoid could not be excluded. CONCLUSION: The majority of HAT antibodies react with a PF4/heparin complex, but there is strong evidence that other antigens are involved in some patients. The HIPA test and the PF4/heparin ELISA are sensitive for diagnosing HAT, and they complement one another.

Enzyme-Linked Immunosorbent Assay↗
PubMed1994

The protein tyrosine kinase pp60c-src is activated upon platelet stimulation.

Stimulation of human platelets causes a dramatic increase in phosphorylation of various proteins at tyrosine residues. The abundance of protein tyrosine kinases of the src-family in platelets, particularly pp60c-src, suggests an important role of these kinases in response to stimulation events. We have shown that pp60c-src is activated on agonist-induced platelet stimulation with respect to its substrate affinity. This was accompanied by phosphorylation of pp60c-src at Ser-12, a residue which is phosphorylated by PKC. Inhibition of PKC with a specific inhibitor, Ro-31-8220, suppressed thrombin-induced translocation of pp60c-src to the cytoskeleton. On the basis of our data, we suggest that the cytoskeletal association of pp60c-src is dependent on phosphorylation of pp60c-src at Ser-12 by PKC. Phosphorylation at Ser-12 in the membrane-binding domain might be the signal that displaces pp60c-src from the plasma membrane and, accompanied with the increased substrate affinity, facilitates phosphorylation of putative substrates.

Arginine Vasopressin↗
PubMed1994

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen.

Sera of 34 patients with heparin-associated thrombocytopenia (HAT), giving a positive result in the serotonin release assay (SRA), were assessed in a platelet factor 4 (PF4)/heparin ELISA. Three sera revealing indeterminate results in the SRA and 10 control sera were also investigated. Both tests correlated closely (Kappa 0.742; p = 2.67 x 10(-7)), but one positive serum in the SRA was negative in the pF4/heparin ELISA. We have isolated the HAT antibodies by absorbtion and elution of HAT sera using endothelial cells (HUVEC). Eluates gave similar results as the sera in the PF4/heparin ELISA (Kappa 0.837, p = 9.26 x 10(-9)), and they also correlated very closely with the SRA (Kappa 0.888; p = 8.89 x 10(-10)). This demonstrates that HAT antibodies bind to the same epitope on platelets and on endothelial cells. High heparin concentrations released PF4 in a dose dependent manner from microtiter plates if PF4/heparin, but not if PF4 alone, was covalently linked. Concomitant to the release of PF4, binding of HAT antibodies to PF4/heparin decreased, as indicated by the median optical density (OD) values of OD 0.88 in the presence of buffer compared to OD 0.181 in the presence of 100 IU/ml heparin. The latter values were similar to those obtained when plates were coated with PF4 alone (median OD 0.203). Binding of three eluates was not inhibited by high heparin concentrations and they reacted also with PF4 alone. We conclude that multimolecular PF4/heparin complexes represent the major antigen in HAT. These multimolecular complexes might present several epitopes and form immune complexes after HAT antibody binding which activate platelets via the FcRII. In a few cases, PF4 alone can be recognized by the antibody. However, there is also evidence that other molecules might be involved in some patients.

Antibodies↗
PubMed1994

[Fc receptor-dependent platelet activation results independently of glycoprotein complex IIb/IIIa].

Platelets of a patient with Glanzmann's thrombasthenia revealed the same activation pattern when stimulated with antibodies of patients with heparin-associated thrombocytopenia (HAT) or immune complexes. This was investigated by the 14C-serotonin release test and by changes in phosphorylation of p20 and p47. Platelet activation by HAT antibodies was completely inhibited by a moab against the platelet FcRII (IV. 3) and by Fc fragments of human IgG but not by F(ab)2 fragments. We conclude that platelet activation via the FcRII occurs independently of the glycoprotein complex IIb/IIIa. Therapeutical approaches targeting GP IIb/IIIa-fibrinogen interaction seem to be not appropriate in HAT.

Blood Platelets↗
PubMed1994

[Suppression of FcRII by intravenously administerd IgG is dependent of preparation procedures of immunoglobulins].

The effect of different i.v. IgG preparations on FcRII-dependent platelet activation by antibodies from patients with heparin-associated thrombocytopenia (HAT) was investigated. Only one preparation (treated with pH 4 and traces of pepsin) sufficiently inhibited FcRII-dependent platelet activation (20 mg/ml). We conclude that administration of high-dose i.v. IgG is efficient for acute treatment of HAT, dependent on the manufacturing process.

Heparin↗
PubMed1993

Pregnancy complicated by heparin associated thrombocytopenia: management by a prospectively in vitro selected heparinoid (Org 10172).

A pregnant woman treated with unfractionated heparin for pelvic vein thrombosis in the 26th week of her first pregnancy developed heparin-associated thrombocytopenia. Diagnosis was verified by the heparin induced platelet activation (HIPA) assay, which revealed cross reactivity to various LMW heparins, but not to the LMW heparinoid Org 10172. Upon intravenous (i.v.) anticoagulation with the heparinoid Org 10172 the platelet count returned to normal within 6 days and remained stable throughout the entire treatment period. After 3 weeks i.v. treatment with Org 10172, administration was changed to the subcutaneous route. At term a healthy boy was delivered spontaneously. No Org 10172 was detected in the cord blood, while therapeutic levels were measured in the maternal blood. The infant's platelet count was normal, but serum from the cord blood induced platelet activation in the presence of heparin, indicating the transplacental passage of heparin-dependent maternal antibodies.

Adult↗
PubMed1993

[Septic-toxic heart failure as a result of a heparin-induced thrombocytopenia with "white-clot-syndrome"].

A patient is presented who developed pulmonary artery embolism and thrombocytopenia with various arterial clots several days after an ankle joint osteosynthesis, which finally caused the amputation of the right arm. Unfractionated heparin was applied for the prevention of postoperative thrombosis. Obviously as a consequence of the liberation of toxic metabolites from the gangrenous lower arm, the patient developed septic shock which was exacerbated by a severe pneumonia caused by methicillin-resistant S. aureus bacteria (MRSA). Laboratory investigations showed an antibody (IgG) induced thrombopenia mediated by all low molecular weight and unfractionated heparins. By eliminating heparin and further treatment with a low molecular weight heparinoid (ORG 10,172), platelet count rose sufficiently and thrombotic process ceased completely. Septic shock was successfully treated by fluid replacement, antibiotics and inotropic substances.

Adult↗
PubMed1993

Heparin-associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides.

The interaction of sulphated oligosaccharides (SO) with platelets and the antibody of heparin-associated thrombocytopenia (HAT type II) was investigated. 3H-heparin binding to platelets was inhibited by different SO, depending on their grade of sulphation. Dextran sulphate, pentosan polysulphate, and heparin were more effective than were LMW heparins. De-N-sulphated heparin and a LMW heparinoid (Org 10172) had no effect. Platelets preincubated with high-grade SO and washed, released serotonin in the presence of HAT sera without additional heparin. Platelets preincubated with HAT sera and then washed were not activated when heparin was added. Only high-grade SO which inhibited heparin binding to platelets caused platelet activation with HAT sera. However, low- and high-grade SO in high concentrations (0.11 g/l) inhibited serotonin release induced by HAT sera and heparin. 32P-phosphorylation of platelet proteins was enhanced by HAT-IgG and heparin and by heat-aggregated IgG, and was inhibited by the moab IV.3. High SO concentrations inhibited only the effect of HAT-IgG and not that of aggregated IgG. We assume that the antigen in HAT involves a releasable platelet protein with a binding site for SO. This was corroborated by studies with an anti-platelet factor 4 antibody causing Fc-receptor dependent platelet activation inhibitable by high SO concentrations.

Antigen-Antibody Complex↗
PubMed1993

Heparin-associated thrombocytopenia: no association of immune response with HLA.

Heparin-associated thrombocytopenia (HAT) is a severe adverse reaction of heparin therapy. Patients with the immunologic type of HAT are at risk of developing arterial and venous thromboembolic complications caused by an antibody which activates platelets in the presence of heparin. Yet, there are no means to identify patients at risk of developing HAT before heparin administration. We investigated the frequency of HLA class-I and class-II antigens in 47 patients with the immunologic type of HAT verified by a positive two-point heparin-induced platelet activation assay. Compared to a control group of 629 healthy individuals HLA antigens B8 and DR3 were less frequent, whereas DR4 and DR53 had a higher frequency. However, none of these antigens proved to be statistically significant. This is another example of an immunologically mediated disease showing no discernible association with the HLA system.

Adult↗
PubMed1993

[Bernard-Soulier syndrome. An important differential diagnosis in chronic thrombocytopenia with bleeding complications].

Hereditary types of thrombocytopenia are often clinically misdiagnosed as chronic autoimmune thrombocytopenia. The Bernard-Soulier syndrome is a recessively inherited thrombocytopenia and -pathy associated with giant platelets. Bleeding time is usually prolonged, hemorrhage can be severe. In a Turkish thrombocytopenic boy autoimmune thrombocytopenia was diagnosed at the age of 11 months because the platelet count raised from 40.000/microliter to 100.000/microliters under treatment with steroids. The correct diagnosis of Bernard-Souiler syndrome was made at the age of 2 1/2 years. His also affected 13 year old sister had been treated for chronic autoimmune thrombocytopenia for years and suffered from severe hypermenorrhoea. Family history revealed consanguinity of the parents. Diagnosis of Bernard-Soulier syndrome was made by crossimmunoelectrophoresis and immunofluorescence showing absence of the glycoprotein complex Ib/IX.

Adolescent↗
PubMed1992

Heparin-associated thrombocytopenia: the antibody is not heparin specific.

In this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Heparin-associated thrombocytopenia: successful therapy with the heparinoid Org 10172 in a patient showing cross-reaction to LMW heparins.

A patient suffering from heparin-associated thrombocytopenia (HAT), recurrent arteriothromboses, and acute renal failure after treatment with standard heparin is described. He failed to improve when therapy was continued with low-molecular-weight (LMW) heparin (Fragmin, Kabi Pfrimmer, Erlangen, FRG). By means of the in vitro heparin-induced platelet activation (HIPA) assay it was shown that standard heparin and the LMW heparins Fragmin and Fraxiparin (Sanofi Labaz, Munich, FRG), as well as the enoxaparine Clexane (Nattermann, Cologne, FRG), all induced platelet activation with the patient's serum. In contrast, the LMW heparinoid Org 10172 (Organon, Oss, The Netherlands) did not cause platelet activation. When the patient was subsequently treated by parenteral administration of Org 10172 as anticoagulant over a period of several weeks the number of platelets rapidly increased and the patient almost completely recovered. This case shows that strong in vivo and in vitro cross-reactivity between standard heparin and LMW heparins may occur, but can be avoided by the use of a novel heparinoid, Org 10172. The HIPA assay provides a simple and sensitive laboratory method for the choice of an innocuous heparin or heparinoid for continued parenteral anticoagulation.

Aged↗