A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes--platelet suspensions stimulated by phorbol diester.

Mononuclear leukocytes release an inhibitor of plasminogen activators. Mononuclear leukocyte mixtures (400 to 1,000/mm3) lysed fibrin (8.3 microM) clots in the presence of plasminogen (0.58 microM). Anti-urokinase IgG (0.16 microM) inhibited this fibrinolysis. 2-Deoxyglucose (5 mM) and oligomycin (2.3 microM) also inhibited fibrinolysis. Incubation of mononuclear leukocytes (3,200/microliter) with phorbol-12 myristate 13-acetate (20 nM) for ten minutes at 37 degrees C aggregated the monocyte and platelet components and inhibited fibrinolysis. The releasate from these stimulated cells in dilutions ranging from undiluted to 1:16 inhibited urokinase (1.6 pM) and tissue plasminogen activator (1.4 pM). This releasate did not inhibit plasmin (2.5 nM). Incubation of this releasate with activated protein C (33 nM to 333 nM) for ten minutes at 37 degrees C before addition of either urokinase, or tissue plasminogen activator and plasminogen completely prevented this inhibition. Thrombin, factor Xa, DIP-activated protein C had no affect on this inhibition. We conclude that activated protein C facilitates fibrinolysis by preventing inhibition of plasminogen activators. This may be a mechanism by which activated protein C increases fibrinolytic activity in vivo.

Whole blood clot lysis: in vitro modulation by activated protein C.

Lysis of clots prepared from native or citrated whole blood as measured by release of 125I fibrinogen degradation products was 10% or less at 20 hours. Lysis of these clots was accelerated by activated protein C in a dose-dependent manner (0.1 to 20 micrograms/ml) from less than 10% to 60-80% at 20 hours. Lysis of clots prepared from native or citrated platelet poor plasma across the same concentration range of activated protein C was less than 15%. Gla-domain-less activated protein C was equally effective in accelerating clot lysis whereas DIP-activated protein C or factor Xa did not accelerate clot lysis. This suggested that this action of activated protein C was enzymatic and this this action was limited to protein C among the vitamin K dependent proteins. The unresponsiveness of platelet poor plasma to activated protein C was completely restored to that of whole blood by addition of mononuclear leukocytes. Addition of red corpuscles or platelets alone had no effect on this response, while addition of polymorphonuclear leukocytes partially restored this response. Addition of metabolic inhibitors 2-deoxyglucose and oligomycin inhibited the response of whole blood and of plasma-mononuclear leukocytes to activated protein C. Reconstitution studies of platelet poor plasma made deficient in plasminogen activator and plasminogen showed that accelerated clot lysis produced by mononuclear leukocytes and activated protein C required the presence of plasminogen. We concluded, therefore, that activated protein C accelerates whole blood or plasma-leukocyte clot lysis by modulating activation of the plasminogen system by metabolically active leukocytes.

Observations on optimal conditions for lysis of whole blood clots and use of this assay as a screening assay in clinical investigation.

These studies describe an assay of whole blood clot lysis as measured by release of 125I-fibrinogen degradation products. Optimal rates of lysis were obtained at 37 degrees C in 10-12 mM EDTA or 3,8% citrate and 4 u of thrombin/ml. Eighteen normal subjects and eight patients (six with recurrent deep vein thrombosis, one with thrombasthenia, and one with hepatitis and resolving portal vein thrombosis) were studied using this assay. The clots of seventeen of the eighteen normal subjects were 50% lysed at 40 hours. The clots of the patients with venous thrombosis and thrombasthenia did not lyse whereas the clots of the patient with hepatitis, resolving portal vein thrombosis and a high plasminogen activator level (0.32 CTA units) were 100% lysed at 4.5 hrs.

Evaluation of soluble CD44 in patients with breast and colorectal carcinomas and non-Hodgkin's lymphoma.

CD44 is a transmembrane glycoprotein involved in cell-cell and cell-substrate interactions. As a cell surface molecule, CD44 may be shed or released into the circulation by proteolytic enzymatic mechanisms. Therefore, soluble CD44 can be found in cell culture supernatants as well as in plasma. In this study we evaluated the levels of soluble total CD44 (sCD44) in serum samples of patients with breast and colorectal carcinoma as well as non-Hodgkin's lymphoma in order to correlate prognosis with sCD44 expression. Besides, we evaluated other clinical tumour markers routinely used, Cancer Antigen (CA) 15.3 and CA 19.9. We investigated 132 serological samples from breast cancer patients, 48 sera from colorectal tumours, 48 samples from stage IV non-Hodgkin's lymphoma and sera from 80 individuals without evidence of cancer or autoimmune disease. Breast cancer patients were divided into three groups: a) patients with no clinical evidence of positive nodules and no metastatic disease; b) patients with positive nodules; and c) patients with metastasis. sCD44 mean serum levels in these groups were 198+/-54 ng/ml, 221+/-78 ng/ml and 242+/-119 ng/ml, respectively, while the marker CA 15.3 values were 15.6+/-6.6 U/ml, 14.0+/-5.8 U/ml and 211.5+/-358.9 U/ml, respectively. sCD44 levels for colorectal tumour were 243+/-72 ng/ml, while CA 19.9 serum levels were 230+/-270 U/ml. Stage IV non-Hodgkin's lymphoma sCD44 levels were 398+/-160 ng/ml. sCD44, CA 15.3 and CA 19.9 values for healthy individuals without evidence of any cancer pathology were 223+/-58 ng/ml, 16.4+/-6.2 U/ml and 33+/-14 U/ml, respectively. From these results we conclude that sCD44 might be used as a reliable marker for patients with non-Hodgkin's lymphoma. However, sCD44 levels failed to correlate with prognosis, tumour burden or metastasis in breast and colorectal cancer patients. Neither was any correlation found between high CA 15.3 or CA 19.9 levels and soluble CD44 serum level.

Alternative exon-specific PCR method for the analysis of human CD44 isoform expression.

CD44 is a widely expressed cell-surface transmembrane glycoprotein involved in diverse adhesive processes. Its isoforms have been implicated in tumor progression and are considered a promising marker for evaluation of the metastatic potential of various tumors. Several methods have been described for the analysis of CD44 isoforms in tumor cells, including immuno-histochemistry, RT-PCR followed by hybridization and nested RT-PCR. We describe an alternative nested PCR for the analysis of CD44 isoform expression in various malignancies. Total RNA was isolated from various shock-frozen tissues from human tumors, reverse-transcribed and PCR-amplified using CD44-specific primers. Reverse-transcription was performed by two different methods, either using Tth-polymerase or MMLV-RT. Exon-specific amplification was then carried out using specific primers for each variable exon. Amplification products were assayed by agarose gel electrophoresis. Comparison of the patterns obtained from the first amplification and from the exon-specific amplification allowed to identify exons expressed by tumor tissues, as well as the genomic organization of CD44 isoforms. The method developed proved to be sensitive, reliable and inexpensive in comparison with other methods. It can be performed even in solid tumors and for numerous samples, and is suitable for laboratories with limited resources.

Induction of apoptosis in murine lymphoma cells by cyclosporin A.

The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

Objective assessment of voice intervention: a clinical audit.

This clinical audit arose from research (Lockhart et al. 1997) which suggested timescales for the care episodes of diagnostically related groups of dysphonic clients, where SLT was directed at targeting the parameters of voice production most requiring change. The objectives of the audit were to examine current practice, compare it with the standards set and with the research findings, address any issues highlighted with regard to service delivery or staff training and to provide information to management on outcomes of care.

Role of platelets in lysis of dilute plasma clots: requirement for metabolically active platelets.

The role of platelets in clot lysis has been investigated functionally with the use of dPRP clots formed at 4 degrees and shifted to 37 degrees. Clots handled in this manner lysed in 6 hr (+/- 1 hr), whereas clots formed at 4 degrees or at 37 degrees and held at those temperatures, or clots formed from dPPP did not lyse in less than 20 hr. dPRP clots having the shorter (6 hr) lysis time released 14C-5-HT at the time of the temperature shift. Preincubation of dPRP with antimycin A and 2-deoxy-D-glucose before addition of thrombin prolonged the clot lysis to 26 hr and inhibited release of 14C-5-HT at the time of the temperature shift. These studies demonstrate that metabolically active platelets are required to mediate the optimal clot lysis seen in the 4 degrees to 37 degrees system and that they continue to function (i.e., take up and release 5-hydroxytryptamine) after they have been incorporated into a clot. Thus the dPRP clot lysis system provides a model by which the timing and sequence of the interaction of metabolically active platelets with the fibrin framework of the formed clot can be studied.

Effect of crosslinking on the structure of solubilized fibrin degradation products in whole plasma.

The purpose of these studies was to establish the validity of 125I fibrin autoradiography--SDS gel techniques for monitoring degradation products from whole plasma or blood clots. These methods can be used to study fibrin degradation not only in patients with congenital factor XIII deficiency, but also in patients with disseminated intravascular coagulation or deep vein thrombosis during the course of thrombolytic therapy. Such an assay might complement existing immunologic techniques to characterize fibrin degradation in vivo by providing an in vitro analysis of the rate and pattern of fibrin degradation in whole blood or plasma. Fibrin degradation was traced by Coomassie blue staining for protein and by autoradiography on SDS-PAGE of degradation products released from a 125I-labeled fibrin tracer. The degradation of non-crosslinked clots from purified fibrin supplemented with plasmin showed a typical release of X, Y, D, and E fibrin fragments. Subsequently, all X and Y fragments were digested to D and E fragments. The degradation of non-crosslinked washed clots prepared from plasma supplemented with plasmin reflected the same pattern. The degradation of non-crosslinked washed clots prepared from EDTA anticoagulated plasma without added plasmin also showed release of X, Y, D, and E fragments. However, in contrast to the non-crosslinked washed clots supplemented with plasmin, there was no additional degradation of the X and Y fragments. These studies established that the pattern of degradation of the 125I-radiolabeled fibrin tracer was similar to that of the total protein released from the fibrin clot as observed by protein staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets.

Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated. The releasate of gel-filtered human platelets stimulated with thrombin served as a source of PAI. When 6 X 10(8) platelets/mL were incubated with thrombin (1 IU/mL), the releasate contained 18 to 26 ng/mL PAI as determined by incubation of the releasate with urokinase and measurement of residual urokinase activity on plasminogen (S2251). Preincubation of PAI with up to 4 micrograms/mL APC for two hours yielded less than 20% neutralization of PAI activity. In the presence of protein S, phospholipid, and Ca2+, neutralization of PAI activity was time-dependent with 50% neutralization occurring in two hours with 1 microgram/mL APC. The cofactor effects of protein S and phospholipid were concentration-dependent with half-maximal acceleration at approximately 3 micrograms/mL protein S and 10 micrograms/mL phospholipid when the experiments were performed at 1 microgram/mL APC. Diisopropylfluorophosphate-inactivated APC, gla-domainless APC, and thrombin-cleaved protein S had no effect on PAI activity, indicating requirement for preservation of the APC active site and of the Ca2+ binding ability of both APC and protein S. These results suggest coordinate binding of APC and protein S onto phospholipid membrane as a prerequisite for optimal expression of PAI neutralized by APC.

Endothelial cell protein C receptor plays an important role in protein C activation in vivo.

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

Role of free protein S and C4b binding protein in regulating the coagulant response to Escherichia coli.

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S. The second objective was to examine the relevance of this model to the hemolytic uremic syndrome. Infusion of C4b-binding protein alone reduced free protein S and decreased platelet concentration to 20% of baseline, whereas infusion of the C4b-binding protein/protein S complex did not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a transient inflammatory response with no reduction of free protein S. However, coinfusion of C4b-binding protein with sublethal E coli reduced free protein S and produced a thrombocytopenia, anemia, and a microvascular thrombotic response, whereas infusion of the C4b-binding protein/protein S complex with sublethal E coli did not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing (S-145) antibodies with protein S coinfused with sublethal E coli produced similar contrasting results. Therefore, we concluded that neutralization of free protein S, and not some other property of C4b-binding protein influenced by protein S, accounted for this microvascular thrombotic response. This response is similar to the hemolytic uremic syndrome characterized by thrombocytopenia, anemia, shistocytosis, and renal glomerular thrombosis with uremia. Comparison of the respective renal histopathologic appearance supports this conclusion. This raises the possibility that inhibition of protein S activity (possibly by one of the forms of C4b-binding proteins) might be one of the factors contributing to microvascular thrombotic disorder, such as the hemolytic uremic syndrome.

DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage.

One of the aims of research in the area of thrombosis has been to design an effective anticoagulant that would function in a predictable and direct manner. In evaluating the role of coagulation in sepsis we used factor Xa blocked in the active center with [5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl+ ++ chloromethyl ketone (DEGR-Xa). We infused 1 mg/kg of DEGR-Xa together with LD100 concentrations of Escherichia coli (4 x 10(10) organisms/kg) into five baboons. As controls, we infused E coli alone into five baboons. The inflammatory, coagulant, and cell injury responses to E coli of both the treated and control groups were lethal and were similar in every respect except for the complete inhibition of the consumption of fibrinogen in the DEGR-Xa group. The half life of DEGR-Xa was approximately 10 hours and 2 hours, as determined by isotopic and enzyme-linked immunosorbent assays, respectively. These results for the first time demonstrate that, although coagulation occurs in E coli sepsis, fibrin formation per se did not influence the lethal outcome in this model. These results also show the effectiveness of DEGR-Xa as an anticoagulant and raise the possibility that it could serve as an alternative to anticoagulants currently in use.

Dysfunction of endothelial protein C activation in severe meningococcal sepsis.

BACKGROUND: Impairment of the protein C anticoagulation pathway is critical to the thrombosis associated with sepsis and to the development of purpura fulminans in meningococcemia. We studied the expression of thrombomodulin and the endothelial protein C receptor in the dermal microvasculature of children with severe meningococcemia and purpuric or petechial lesions. METHODS: We assessed the integrity of the endothelium and the expression of thrombomodulin and the endothelial protein C receptor in biopsy specimens of purpuric lesions from 21 children with meningococcal sepsis (median age, 41 months), as compared with control skin-biopsy specimens. RESULTS: The expression of endothelial thrombomodulin and of the endothelial protein C receptor was lower in the patients with meningococcal sepsis than in the controls, both in vessels with thrombosis and in vessels without thrombosis. On electron microscopical examination, the endothelial cells were generally intact in both thrombosed and nonthrombosed vessels. Plasma thrombomodulin levels in the children with meningococcal sepsis (median, 6.4 ng per liter) were higher than those in the controls (median, 3.6 ng per liter; P=0.002). Plasma levels, protein C antigen, protein S antigen, and antithrombin antigen were lower than those in the controls. In two patients treated with unactivated protein C concentrate, activated protein C was undetectable at the time of admission, and plasma levels remained low. CONCLUSIONS: In severe meningococcal sepsis, protein C activation is impaired, a finding consistent with down-regulation of the endothelial thrombomodulin-endothelial protein C receptor pathway.