C. Korninger

Sandwich ELISA for t-PA antigen employing a monoclonal antibody

A sandwich enzyme-linked immunosorbent assay (ELISA) for determination of tissue-type plasminogen activator (t-PA) was developed. 96-well flat-bottom polystyrene plates were coated with polyclonal (goat) anti t-PA IgG (10 micrograms/ml). After addition of samples monoclonal (murine) anti t-PA IgG (1.5 micrograms/ml) was added. Finally, peroxidase labelled anti-mouse IgG (goat) was used to quantify the bound second antibody. The assay can be used for determination of t-PA antigen in purified systems, in cell culture supernatants, and in human plasma, provided that EDTA (0.005 M) is present in the sample. In 78 healthy volunteers, t-PA antigen levels at rest were 0.4 - 15.2 ng/ml (4.3 +/- 2.7, means +/- S.D.); A significant positive correlation between t-PA antigen and age could be demonstrated.

Activation of coagulation and fibrinolysis in patients with arterio-sclerosis: Relation to localization of vessel disease and risk factors

Activation markers of blood coagulation and fibrinolysis and several fibrinolytic parameters were determined in arteriosclerotic patients to investigate the relation between extension and main localization of vessel disease, risk factors and disturbances within the blood coagulation and the fibrinolytic system. Indications of an increased intravascular fibrin formation and subsequent fibrinolysis were found in peripheral artery disease (PAD) patients but not in coronary artery disease (CAD) patients. Compared with healthy controls PAD patients had elevated TAT (median: 3.2 ng/ml, 1.5-70 vs. 2.1, 1.2-4.7, p less than 0.005) and D-Dimer (median: 365 ng/ml, range 85-2000 vs. 185, 79-360; p less than 0.0001) plasma levels, whereas TAT (2.4, 1.2-13) and D-Dimer (190, 58-1000) levels of CAD patients were in the normal range. No associations were detected between risk factors of arteriosclerosis (hyperlipidemia, diabetes mellitus, cigarette smoking, hypertension) and the plasma levels of the activation markers TAT and D-Dimer. Independent from risk factors PAD and CAD patients had elevated plasma plasminogen activator inhibitor capacity (PAI cap). Our results provide evidence that 1) increased plasma levels of blood coagulation and fibrinolysis activation markers are not related to risk factors of arteriosclerosis but seem to be unspecifically caused by activation processes on arteriosclerotic vessel wall defects, 2) increased plasma PAI cap found in arteriosclerotic patients is a relatively unspecific phenomenon associated with arterial vessel disease.

Hepatic synthesis and clearance of components of the fibrinolytic system in healthy volunteers and in patients with different stages of liver cirrhosis

We determined plasma levels of tissue-type plasminogen activator (t-PA) antigen, urokinase-type plasminogen activator (u-PA) antigen, and activity of the fast acting inhibitor of plasminogen activator (PAI-1) in patients with different stages of liver cirrhosis (Child A, B, and C) and in age and sex-matched healthy controls to investigate the contribution of the liver to the metabolism of these main components of the fibrinolytic system. For control purposes routine clotting parameters were also determined. In patients with the most severe form of liver cirrhosis (Child C) t-PA antigen levels were significantly elevated as compared to patients with Child A or Child B (p less than 0.05) or to controls (p less than 0.01). Furthermore, Child C patients exhibited significantly decreased PAI-1 plasma levels (p less than 0.05) as compared to controls. We were not able to demonstrate, however, any significant correlation between liver function and u-PA plasma levels. Furthermore, t-PA antigen and albumin plasma levels were negatively correlated (r = 0.48; p = 0.0015) and t-PA antigen and bilirubin were positively correlated (r = 0.46; p = 0.0022) thus indicating that the liver is mainly involved in the clearance of t-PA antigen. PAI-1 activity, however, seems to depend partially on synthesis by the liver as demonstrated by a positive correlation between PAI-1 and albumin (r = 0.33; p = 0.037). These physiologic liver functions are both progressively attenuated in severe liver damage and an increase of t-PA plasma levels and a decrease of PAI-1 might contribute to the higher fibrinolytic tendency observed in those patients.

Thromboprophylaxis with 60 mg enoxaparin is safe in hip trauma surgery

BACKGROUND Little information is available concerning dosage and optimal initiation of thromboprophylactic therapy with low-molecular-weight heparin (enoxaparin) in nonelective hip surgery. The aim of our prospective study was to evaluate the incidence of clinically apparent deep vein thrombosis (DVT), pulmonary embolism (PE), and major hemorrhage in patients receiving thromboprophylaxis with enoxaparin undergoing hip surgery after hip fracture. METHOD From 946 consecutive patients admitted with hip fractures, 897 were operated on and received enoxaparin according to the following regimen: Preoperative heparinization from time of admission onwards. Administration of 60 mg enoxaparin, in two doses (20 and 40 mg subcutaneously), during the first 5 days postoperatively. Prophylaxis for a minimum of 5 weeks (40 mg daily). RESULTS Clinical signs of DVT were present in 37 patients (4.2%), who all underwent venography. In five patients, DVT was confirmed (0.6%). None of these patients suffered from PE. Another four patients (0.4%) developed clinical signs of PE, and suspected diagnosis was confirmed by computed tomographic scan in two (0.2%). No deaths because of PE were observed. Major hemorrhage occurred in 42 patients (4.7%), there was one death from hemorrhage caused by an intracerebral event. No case of heparin-induced thrombocytopenia type II was observed. CONCLUSION Thromboprophylaxis with 60 mg enoxaparin daily, in split doses, starting before surgery, is safe and appropriate in patients with hip fractures. Clinically apparent DVT and PE are rarely observed, and bleeding complications are comparable to those occurring with a conventional thromboprophylactic regimen.

Platelet aggregation inhibitors, platelet function testing, and blood loss in hip fracture surgery.

BACKGROUND The purpose of our prospective study was to analyze how many patients with hip fractures are on treatment with platelet aggregation inhibitors (aspirin and clopidogrel), how many of these patients have impaired platelet function as measured by the PFA-100, and whether there is an association between perioperative blood loss and either intake of platelet inhibitors or platelet function. METHODS Four hundred sixty-two patients with hip fractures were investigated. Surgery (most commonly dynamic screw fixation and hemiarthroplasty) was performed on day 1.3 (in patients on clopidogrel on day 3). Platelet function analysis was performed with the PFA-100, using the collagen and epinephrine closure time. Transfusion requirement and drain blood loss were measured. RESULTS Ninety-eight patients (21%) were on treatment with aspirin, of those, 64 patients (65%) had impaired platelet function. Twenty-two patients (5%) were on clopidogrel, of those, 15 patients (68%) had impaired platelet function. Of the patients without platelet aggregation inhibitors, 29% had impaired platelet function. Mortality, major bleeding, red blood cell requirement, and drainage blood loss did not correlate with platelet aggregation inhibitor intake or platelet function. CONCLUSIONS It is not possible to predict the platelet function by asking patients about intake of aspirin or clopidogrel. Perioperative blood loss did not correlate with either history of platelet aggregation inhibitor intake or platelet function as determined by PFA-100. Therefore, the measurement of platelet function is of little clinical relevance in patients with hip fractures. In patients treated with aspirin, surgery should not be delayed, and patients on clopidogrel can be operated on 3 days after stopping the drug without increased bleeding risk.

Disseminated intravascular coagulation and decrease in fibrinogen levels induced by vincristine/prednisolone therapy of lymphoid blast crisis of chronic myeloid leukemia

SummaryTherapy with vincristine (2 mg i.v. weekly) and prednisolone (100 mg p.o. daily) caused a decrease in fibrinogen levels in nine patients treated for lymphoid blast crisis (LBC) of chronic myeloid leukemia (CML). During the first days of treatment disseminated intravascular coagulation (DIC), evidenced by a positive ethanol gelation test, markedly increased thrombin-antithrombin III complex and fibrin-split product D-dimer levels, and a rapid fall in fibrinogen levels was observed in two patients. The induction of DIC in these two patients caused profuse bleeding in one and necessitated substitution therapy with fibrinogen and platelet concentrates. The remaining seven patients revealed no signs of DIC; nevertheless, four of them showed a moderate increase in D-dimer levels after initiation of therapy. In these patients a well-known side effect of long-term steroid therapy, namely a decrease of fibrinogen levels, was observed within the first week of treatment. Fibrinogen levels did not fall below 150 mg/dl and increased after dose reduction from 100 mg/day to 50 mg/day. We conclude from our results that two types of disturbances in fibrinogen metabolism can be observed during vincristine/prednisolone therapy of LBC of CML: (a) a decrease of fibrinogen levels due to a steroid-mediated impairement of liver synthesis, and (b) a rapid fall in fibrinogen levels in the course of DIC, most likely induced by the release of procoagulants from detoriorating blast cells, leading to severe bleeding in selected cases.

The prevalence of hereditary antithrombin-III deficiency in patients with a history of venous thromboembolism

Antithrombin-III activity was determined in 752 patients with a history of venous thrombosis and/or pulmonary embolism. 54 patients (7.18%) had an antithrombin-III activity below the normal range. Among these were 13 patients (1.73%) with proven hereditary deficiency. 14 patients were judged to have probable hereditary antithrombin-III deficiency, because they had a positive family history, but antithrombin-III deficiency could not be verified in other members of the family. In the 27 remaining patients (most of them with only slight deficiency) hereditary antithrombin-III deficiency was unlikely. The prevalence of hereditary antithrombin-III deficiency was higher in patients with recurrent venous thrombosis.

Acquired Deficiency of Fibrinogen (Fbg) and Factor VII (F VII) During and Following Immunosuppression for Severe Aplastic Anemia (SAA) with Antithymocyte Globulin (ATG) and High-Dose Methylprednisolone (MP)

While determing clotting and fibrinolysis proteins by conventional laboratory test kits before, during and after ATG/MP treatment we found a yet undescribed reversible deficiency of Fbg and F VII.