bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Intrinsic and Extrinsic Plasminogen upon the Lysis of Thrombi In Vitro
Authors: Nishino N , Kakkar V V , Scully M F
Published: July 2018

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.
Biochemical Characterization of PECAM-1 (CD31 Antigen) on Human Platelets
Authors: Metzelaar Marcel J , Korteweg Jeanne , Sixma Jan J , Nieuwenhuis H Karel
Published: July 2018

The platelet plasma membrane expresses several membrane glycoproteins with a high molecular weight. In this study we have investigated the properties of the CD31 antigen on platelets and endothelial cells using the monoclonal antibody (MoAb) RUU-PL 7E8. Comparative studies revealed that the CD31 antigen, PECAM-1 and endoCAM are the same protein. The CD31 antigen was immunoprecipitated with a molecular mass of 125 kDa nonreduced and 135 kDa reduced from Nonidet-P40 lysates of surface labeled human platelets. The relative position in two-dimensional nonreduced/reduced SDS-PAGE and IEF-PAGE, compared to other glycoproteins of similar molecular weight, was elucidated. The position of the CD31 antigen was clearly distinct from the position of the platelet membrane glycoproteins Ia, Ib, IIa, IIb, IIIa and the granule membrane protein GMP-140. Native resting platelets bound 7,760 ± 1,670 molecules/platelet, whereas thrombin-stimulated platelets bound 14,500 ± 3,790 molecules/platelet. Immunoelectron microscopy revealed the presence of the CD31 antigen on the membrane of both resting and thrombin-activated platelets. Immunofluorescence studies showed the presence of the CD31 antigen in the membrane of endothelial cells on sites of cell-cell contact, suggesting that the CD31 antigen might be involved in cell-cell interaction. In functional studies, MoAb RUU-PL 7E8 did not inhibit platelet aggregation, platelet adherence to the extracellular matrix of endothelial cells and purified collagen fibrils under flow conditions, nor was any influence found on endothelial cell detachment and growth.
Role of Internalization in Platelet Activation Induced by Collagen Fibers – Differential Effects of Aspirin, Cytochalasin D, and Prostaglandin E1
Authors: Ruf Andreas , Patscheke Heinrich , Morgenstern Eberhard
Published: July 2018

Washed human platelets were stimulated with fibrillar collagen and platelet aggregation was prevented by non-stirring conditions. In these samples, electron microscopy revealed three fractions of platelets: 1) a majority without contacts to the collagen fibers, 2) with focal contacts to collagen, and 3) a small fraction of platelets with internalized collagen. All platelets had undergone shape change, and exhibited an internal contraction and granule release. However, only those with internalized collagen were completely degranulated. The internalized collagen was found to be in close contact to the contractile sphere in the platelet center, as it was demonstrable with computer assisted 3-D reconstruction from serial sections. Aspirin inhibited neither the adhesion to collagen nor its internalization by internal contraction. Also it did not impair the shape change and degranulation in the platelet fractions that internalized collagen. However, aspirin blocked the shape change and internal contraction of the other platelets and inhibited the [3H]serotonin release. Cytochalasin D 0.1 uM also suppressed the internalization of collagen, the shape change, the formation of a contractile gel, the degranulation, and the [3H]serotonin release in all platelets, whereas the number of platelets that adhered to collagen remained unchanged. The same effects were produced by prostaglandin E1. If the platelets were stimulated with the TXA2 mimetic, U46619, cytochalasin D at 0.1 εM had no effect; but at 20 εM it strongly enhanced the degranulation and the [3H]serotonin release, although the platelets remained discoid. It is concluded that collagen triggers a focal activation of an adherent platelet at the site of its initial contact to collagen. This subthreshold activation proceeds to degranulation and TXA2 formation via a feedback cascade which involves internal contraction, internalization of collagen and multiplication of the collagen membrane contacts.
In Vivo Recovery and Survival of Monoclonal-Antibody-Purified Factor VIII Concentrates
Authors: Kasper Carol K , Kim Hugh C , Gomperts Edward D , Smith Kenneth J , Salzman Phyllis M , Tipping Diane , Miller Robert , Montgomery Robert M
Published: July 2018

In response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.
A Rapid and Sensitive Test for Diagnosing Heparin-Associated Thrombocytopenia
Authors: Greinacher A , Michels I , Kiefel V , Mueller-Eckhardt C
Published: July 2018

Heparin-associated thrombocytopenia (HAT) is a severe complication of heparin therapy. Its diagnosis is difficult. Conventional assays employ platelet aggregometry (PAA) and/or 14C-serotonin release (SRA) which are either insensitive (PAA) or require radioactive tracers (SRA). We here describe a newly developed sensitive and rapid assay based on visual evaluation of heparin-induced platelet activation (HIPA) in microtiter wells. Using sera of 34 patients with clinically suspected HAT we found the HIPA assay to be as sensitive as the SRA and superior to PAA. The HIPA assay allows investigation of crossreactivity with different types of heparins, low molecular weight (LMW) heparins and heparinoids. Three patients who required further parenteral anticoagulation and in whom the HIPA assay was negative before treatment with the LMW heparinoid Org 10172, were treated with this new heparinoid without adverse reactions. We conclude that the HIPA assay may be a useful tool for differential diagnosis and therapy in patients with HAT.
Heparin Neutralizing Activity in Patients with Renal Disease on Maintenance Haemodialysis
Authors: Aronstam A , Dennis B , Friesen M J , Clark W F , Linton A L , Lindsay R M
Published: July 2018

Heparin neutralizing activity (HNA) was increased in plasma of patients with end-stage renal disease treated by maintenance haemodialysis. It was not raised in non-dialyzed patients with chronic renal failure, nor in patients with normal renal function who had been exposed to the extracorporeal circulation of the heart-lung bypass 48–72 hr before testing. It is postulated that the trauma of extracorporeal circulation causes platelets to release HNA which is not cleared by the dialysis membranes of the artificial kidney but is by the human kidney. This may have therapeutic implications for heparin dosage schedules during haemodialysis.
Plasmin Digestion of factor VIII: Characterization of the Breakdown Products with Respect to Antigenicity and von Willebrand Activity
Authors: Guisasola J A , Cockburn C G , Hardisty R M
Published: July 2018

Highly purified factor VIII was incubated for up to 24 hours in the presence of plasmin, and the biological activities and peptide structure of the digestion products determined at intervals. Procoagulant activity (VIIIC) was rapidly lost, but 17-32% of the initial ristocetin cofactor (VIIIR: WF) activity remained after 24 hours. Immunoelectrophoresis showed a progressive increase in rocket height and a concomitant increase in electrophoretic mobility of the factor-VIII-related antigen (VIIIR: AG). Crossed immunoelectrophoresis of the 24- hour digest showed three distinct precipitin arcs, of which the major one, with intermediate anodal mobility, gave reactions of non-identity with the other two. On sepharose gel chromatography the 24-hour digest gave three peaks: peak II contained about 80% of the residual VIIIR: WF and resolved on SDS-polyacrylamide gels into a series of peptides with apparent molecular weights between 125,000 and 185,000; these were reduced by mercaptoethanol to fragments of 15,000-80,000 daltons, a 65,000 dalton fragment being particularly strongly PAS positive. We conclude that large molecular size is not a prerequisite for VIIIR :WF activity, and that the presence of factor-VIII breakdown products may be a cause of misleading results in the determination of VIIIR:AG by immunoelectrophoresis.
Calcium-Dependent Activation of Protein C by Thrombin/Thrombomudulin: Role of Negatively Charged Amino Acids within the Activation Peptide of Protein C
Authors: Friedrich Ute , Pötzsch Bernd , Preissner Klaus T , Müller-Berghaus Gert , Ehrlich Hartmut
Published: July 2018

In the absence of its cofactor thrombomodulin (TM) thrombin is only a poor activator of the anticoagulant serine protease protein C (PC). The TM-dependence of PC-activation has been restricted to a series of molecular structures of the PC molecule including high-affinity calcium binding sites and single amino acid residues. However, thrombin induced activation of a PC derivative altered in all these critical positions is markedly enhanced by TM indicating that additional structures of the PC molecule are involved in determining the TM specificity. Based on the hypothesis that such an additional regulatory element should be located near the thrombin cleavage site and should include negatively charged amino acids to ascertain calcium binding, we studied whether Glu and Asp in positions P7 and P6 relative to the thrombin cleavage site together with Asp in P3 are involved in formation of such a regulatory element. Three PC derivatives containing the neutral counterpart of the negatively charged amino acids in positions P3; P3 and P6; and P3, P6, and P7, respectively, were generated using site-directed mutagenesis. Compared to rPC-wt the initial rates of PC activation of all three mutants were increased 4.0-fold for thrombin/TM and 4.0-, 5.3-fold for activation by thrombin alone. However, compared to the PC derivative neutralized exclusively in P3, additional changes in P6 and P7 showed no increase in the thrombin activation kinetics and calcium binding properties were identical in all of the three mutants. We conclude that .1) among the three negatively charged amino acids at the COOH-terminal end of the activation peptide of PC, only Asp in P3 is involved in calcium-dependent inhibition of PC activation by thrombin; 2) the residues in P7 and P6 do not contribute to the calcium binding properties of PC; 3) P7 and P6 sites are not required for calcium-dependent activation of PC by the thrombin/TM complex.
A New Hypothesis: Possible Mechanisms in the Involvement of the Increased Plasminogen Activator Activity in Branching Regions of the Aorta in the Initiation of Atherosclerosis
Authors: Smokovitis A
Published: July 2018

Branching regions of the aorta are predilection regions for atherosclerosis. The intima in the branching regions of the normal aorta shows a constantly increased plasminogen activator activity from early life. At these areas, the endothelium is also damaged, it shows increased permeability, etc. The constantly increased plasminogen activator activity (local plasmin production) might play a protective role or on the contrary might participate in the initiation of atherosclerosis at the branching regions through a number of proved or suggested mechanisms.

No matter what the actual role of the locally increased plasminogen activator activity in the initiation of atherosclerosis is, the role of the fibrinolytic system in the progression of the atherosclerotic lesion seems to be clear. There is an accumulation of evidence that the impaired fibrinolytic activity in atherosclerotics participates in the progression and the complications of the disease.
Acquired von Willebrand’s Syndrome in the Course of Waldenström’s Disease
Authors: Mazurier Claudine , Parquet-Gernez Armelle , Descamps Jacques , Bauters Francis , Goudemand Maurice
Published: July 2018

Acquired von Willebrand's syndrome with a regressive evolution is described in a 66 year old man with Waldenström's disease. An inhibitor electively directed against Ristocetin cofactor activity has been demonstrated, active in vitro after incubation at 37° C. Serum fractionation showed that the inhibitor was independant of the monoclonal IgM and subsequent purification that it was IgG in nature. The results permit its classification as an auto-antibody.
Upper Extremity Deep Venous Thrombosis in Cancer Patients with Venous Access Devices - Prophylaxis with a Low Molecular Weight Heparin (Fragmin)
Authors: Monreal Manuel , Alastrue Antoni , Rull Miquel , Mira Xavier , Muxart Jordi , Rosell Rafael , Abad Albert
Published: July 2018

Central venous access devices are often essential for the administration of chemotherapy to patients with malignancy, but its use has been associated with a number of complications, mainly thrombosis. The true incidence of upper extremity deep vein thrombosis (DVT) in this setting is difficult to estimate since there are very few studies in which DVT diagnosis was based on objective tests, but its sequelae include septic thrombophlebitis, loss of central venous access and pulmonary embolism.

We performed an open, prospective study in which all cancer patients who underwent placement of a long-term Port-a-Cath (Pharmacia Deltec Inc) subclavian venous catheter were randomized to receive or not 2500 IU sc of Fragmin once daily for 90 days. Venography was routinely performed 90 days after catheter insertion, or sooner if DVT symptoms had appeared. Our aims were: 1) to investigate the effectiveness of low doses of Fragmin in preventing catheter-related DVT; and 2) to try to confirm if patients with high platelet counts are at a higher risk to develop subclavian DVT, as previously suggested.

On the recommendation of the Ethics Committee, patient recruitment was terminated earlier than planned: DVT developed in 1/16 patients (6%) taking Fragmin and 8/13 patients (62%) without prophylaxis (Relative Risk 6.75; 95% Cl: 1.05-43.58; p = 0.002, Fisher exact test). No bleeding complications had developed. As for prediction of DVT, there was a tendency towards a higher platelet count in those patients who subsequently developed DVT, but differences failed to reach any statistical significance (286 ±145 vs 207 ±81 X 109/1; p = 0.067). According to our experience, Fragmin at the dosage used proved to be both effective and safe in these patients.
The Effects of Long-term Heparin Therapy During Pregnancy on Bone Density
Authors: Douketis James D , Ginsberg Jeffrey S , Burrows Robert F , Duku Eric K , Webber Colin E , Brill-Edwards Patrick
Published: July 2018

We performed a prospective matched cohort study to investigate the effects of long-term (>1 month) heparin therapy on lumbar spine bone density. Twenty-five women who received heparin during pregnancy, and 25 matched controls underwent dual photon absorptiometry of the lumbar spine in the post-partum period. Zero of 25 heparin-treated patients developed fractures. Heparin-treated patients had a 0.082 g/cm2 lower bone density compared to untreated controls, which is clinically and statistically significant (p = 0.0077). There were 6 matched pairs in which only the heparin-treated patient had a bone density below 1.0 g/ cm2, compared to only one pair in which only the control patient had a bone density below this level (p = 0.089). The correlation coefficients of the difference .in bone density in each matched pair, and the duration of heparin therapy, the mean daily dose, and the total dose of heparin were 0.042, -0.015, and 0.021, respectively; none of these values is statistically significant. We conclude: 1) long-term heparin therapy was associated with a significant reduction in bone density, although fractures are uncommon, 2) there was no significant correlation between lumbar bone density and the dose or duration of heparin.
Elevated Levels of Prothrombin Activation Fragment 1+2 in Plasma from Patients with Heterozygous Arg506 to Gin Mutation in the Factor V Gene (APC-Resistance) and/or Inherited Protein S Deficiency
Authors: Zöller Benget , Holm Johan , Svensson Peter , Dahlbäck Björn
Published: July 2018

Inherited resistance to activated protein C (APC-resistance), caused by a point mutation in the factor V gene leading to replacement of Arg(R)506 with a Gin (Q), and inherited protein S deficiency are associated with functional impairment of the protein C anticoagulant system, yielding lifelong hypercoagulability and increased risk of thrombosis. APC-resistance is often an additional genetic risk factor in thrombosis-prone protein S deficient families. The plasma concentration of prothrombin fragment 1+2 (F1+2), which is a marker of hyper-coagulable states, was measured in 205 members of 34 thrombosis-prone families harbouring the Arg506 to Gin mutation (APC-resistance) and/or inherited protein S deficiency. The plasma concentration of F1+2 was significantly higher both in 38 individuals carrying the FV:Q506 mutation in heterozygous state (1.7 ± 0.7 nM; mean ± SD) and in 48 protein S deficient cases (1.9 ± 0.9 nM), than in 100 unaffected relatives (1.3 ±0.5 nM). Warfarin therapy decreased the F1+2 levels, even in those four patients who had combined defects (0.5 ± 0.3 nM). Our results agree with the hypothesis that individuals with APC-resistance or protein S deficiency have an imbalance between pro- and anti-coagulant forces leading to increased thrombin generation and a hypercoagulable state.
Plasma Ascorbate Protects Coagulation Factors against Photooxidation
Authors: Parkkinen Jaakko , Vääränen Outi , Vahtera Elina
Published: July 2018

It has been suggested that proteins, unlike lipids, are not protected against oxidative damage by antioxidants in plasma. We have studied the effect of photodynamic virus inactivation treatment of fresh human plasma on coagulation factor activities. Photodynamic treatment generates singlet oxygen which causes inactivation of fibrinogen and factor VIII. Other coagulation factors or anticoagulant proteins are clearly less affected. We found that there is an inverse correlation between the extent of coagulation factor inactivation during the treatment and the plasma ascorbate concentration. The inactivation of coagulation factors was prevented in a dose-dependent manner by adding ascorbate to the plasma before the treatment. Ascorbate was consumed during the treatment at an apparently linear rate. Oxidation of urate and coagulation factors was enhanced when ascorbate had disappeared from plasma. These results indicate that ascorbate is a primary antioxidant against photooxidation in plasma and that it effectively prevents oxidative damage to coagulation factors and other proteins.
Markers of Hemostatic System Activation during Thromboprophylaxis with Recombinant Hirudin in Total Hip Replacement
Authors: Cofrancesco E , Cortellaro M , Leonardi P , Corradi A , Ravasi F , Bertocchi F
Published: July 2018

Coagulation activation markers were studied in 148 patients undergoing total hip replacement under recombinant-hirudin (Desirudin, TMRevasc) prophylaxis with the aim of investigating the efficacy and safety of this anticoagulant compared with heparin in terms of biological effects on coagulation variables and bleeding. Hirudin (10, 15 or 20 mg s.c. b.i.d.) or unfractionated heparin (5000IU s.c. t.i.d.) was administered immediately before surgery and continued for 8-12 days. Activated partial thromboplastin time (aPTT), prothrombin activation fragment F1+2 (F1+2), thrombin-antithrombin III complexes (TAT) and D-dimer were measured at baseline and on postoperative days 1,3 and 6, immediately before the morning injection.

In comparison with baseline values, heparin had little effect on aPTT whereas the three hirudin doses prolonged aPTT significantly with no differences among the three doses. Moreover, there were no group differences in perioperative or cumulative blood loss or transfusion requirements. F1+2 fragment, TAT and D-dimer plasma levels were higher than at baseline during the entire postoperative period, with different trends (F1+2 increasing, TAT decreasing, D-dimer increasing, decreasing and then increasing again), but without significant differences among the four treatment groups. Our findings suggest that specific inhibition of thrombin seems a safe and efficacious mode of blocking thrombin activity after hip surgery although it does not prevent thrombin generation.
Evaluation of a New Rapid Quantitative D-dimer Assay in Patients with Clinically Suspected Deep Vein Thrombosis
Authors: D’Angelo Armando , D’Alessandro Gabriella , Tomassini Loredana , Pittet Jean Louis , Dupuy G , Crippa Luciano
Published: July 2018

The sensitivity and specificity for deep vein thrombosis (DVT) of a new rapid, quantitative and precise (total imprecision < 10%) D-dimer assay suitable for individual measurements (VIDAS D-DIMER, bio-Merieux, France) were evaluated in a consecutive series of 103 in- and out-patients submitted to serial compression ultrasonography (C-US) for the clinical suspicion of DVT (n = 66) or of DVT recurrence (n = 37) and symptoms lasting from 1 to 15 days. DVT was found in 22 patients at baseline testing and no patient with an initially negative C-US developed vein incompressibility at follow up. The time elapsed from the onset of symptoms was negatively associated with D-dimer levels both in patients with and in those without DVT. In the entire series of patients, the sensitivity of a positive D-dimer test (≥1.0 Μg/ml) for the presence of DVT was 96% (21/22 patients, 95% confidence interval 75-100%) with a specificity of 75% (64-84%), a negative predictive value of 98% (90-100%), a positive predictive value of 51% (35-67%), and an overall accuracy of 80% (70-87%). A normal D-dimer value (0.22 Μg/ml) was observed in one patient with DVT and symptoms lasting from 15 days. The approach of withholding C-US testing in patients with symptoms lasting from less than 11 days and D-dimer levels below the cut-off value was compared to serial C-US testing alone in a cost-effectiveness analysis subdividing the 66 patients with a first episode according to their clinical pretest probability of DVT. Thrombosis was detected in 6.7% of the patients in the low probability group (n = 15), 16.7% of the patients in the moderate probability group (n = 24), 51.9% of the patients in the high probability group (n = 27) and 8.1% of patients with suspected DVT recurrence. Calculated cost-savings for each DVT diagnosed ranged from 5% in the high pretest probability group to 55% in the low pretest probability group and to 77% in patients with suspected DVT recurrence.

The safety of avoiding C-US testing in symptomatic patients with a negative D-dimer test should be evaluated in clinical management studies.
DDAVP Reduces Bleeding during Continued Hirudin Administration in the Rabbit
Authors: Bove Christina M , Casey Brenda , Marder Victor J
Published: July 2018

Hirudin is a potent thrombin inhibitor derived from the leech Hirudo medicinalis salivary gland which has considerable potential for therapeutic use in thrombotic disease. The major risk attendant its use is hemorrhage. This study investigates the hypothesis that the prohemostatic effects of DDAVP infusion can curtail the hemorrhagic effect induced by ongoing hirudin administration.

In a randomized and blinded manner, rabbits were exposed to a 15-min intravenous infusion of DDAVP or saline midway through a continuous two-h intravenous infusion of hirudin. Bleeding time was monitored by full thickness ear punctures performed before, during and after hirudin exposure.

Hirudin induced a significant hemorrhagic state, manifest as a 7-10-fold prolongation of the primary bleeding time. DDAVP reduced the mean duration of primary bleeding from 10.8 min to 5.9 min (p = 0.001) as well as the number of sites which bled for longer than 6 or 20 min (46% vs 27%, p = 0.002; and 18% vs 5%, p = 0.002, respectively). Although there was no difference in the incidence of spontaneous rebleeding from these sites (44 vs 36%, p = 0.21), rebleeding did not persist as long in animals that received DDAVP (8 vs 16 min, p = 0.005), and fewer sites rebled for longer than 20 min (8 vs 27%, p = 0.027). Results were essentially the same for two different commercial recombinant hirudin preparations.

DDAVP appears to attenuate the bleeding caused by continuous hirudin infusion in rabbits and establishes a foundation for clinical assessment in patients.
The Relationship between Plasma Microparticles, Protein S and Anticardiolipin Antibodies in Patients with Human Immunodeficiency Virus Infection
Authors: Gris Jean-Christophe , Toulon Pierre , Brun Sophie , Maugard Claude , Sarlat Christian , Schved Jean-François , Berlan Jocques
Published: July 2018

The high prevalence of free protein S deficiency in human immunodeficiency virus (HlV)-infected patients is poorly understood. We studied 38 HIV seropositive patients. Free protein S antigen values assayed using the polyethylene-glycol precipitation technique (PEG-fS) were statistically lower in patients than in controls. These values using a specific monoclonal antibody-based ELISA (MoAb-fS) and the values of protein S activity (S-act) were not statistically different between patients and controls. C4b-binding protein values were not different from control values. In patients, PEG-fS values were lower than MoAb-fS values. Ten patients had a PEG-fS deficiency, 4 patients had a MoAb-fS deficiency and 8 had a S-act deficiency. Protein S activity and MoAb-fS were lower in clinical groups with poor prognosis and in patients with AIDS but PEG-fS was not. A trend for reduced S-act/MoAb-fS ratios was observed in patients. PEG-fS was negatively correlated with anticardiolipin antibody titers whereas MoAb-fS was not. The plasma of PEG-fS deficient HIV-patients contained high amounts of flow cytometry detectable microparticles which were depleted from plasma by PEG precipitation. The microparticles were partly CD42b and CD4 positive but CD8 negative. These microparticles were labelled by an anti free protein S monoclonal antibody. The observed differences between MoAb-fS and PEG-fS values were correlated with the amount of detectable plasma microparticles, just like the differences between MoAb-fS and S-act. Plasma microparticles correlated with anticardiolipin antibody titers.

In summary, free protein S antigen in HIV infected patients is underestimated when the PEG precipitation technique is used due to the presence of elevated levels of microparticles that bind protein S. The activity of free protein S is also impaired by high levels of microparticles. The prevalence of free protein S deficiency in HIV positive patients is lower than previously published (4/38, -10%) and is correlated with poor prognosis. By implication, use of a PEG precipitation technique might give artefactually low free protein S antigen values in other patient groups if high numbers of microparticles are present. In HIV patients, high titers of anticardiolipin antibodies are associated with high concentrations of cell-derived plasma microparticles.
Differential Effects of Warfarin on the Intracellular Processing of Vitamin K-dependent Proteins
Authors: Wu Wei , Bancroft John D , Suttie J W
Published: July 2018

The vitamin K-dependent carboxylation of specific glutamyl residues to γ-carboxyglutamyl residues occurs during the endoplasmic reticulum processing of a limited number of proteins. The fate of the under-γ-carboxylated proteins during protein processing was studied. When human hepatoma (HepG2) cells were grown in the presence of warfarin, under-γ-carboxylated prothrombin was secreted into the medium. In contrast, prothrombin secretion from a rat hepatoma (H-35) cell line was blocked by warfarin, and intracellular forms which were retained were degraded. When rat prothrombin (rFII) was stably transfected into warfarin treated HepG2 cells, endogenous human prothrombin (hFII) was secreted in an under-γ-carboxylated form, while rFII accumulated intracellularly. These data indicate that retention and degradation of under-γ-carboxylated prothrombin by human hepato-cytes is related to a structural difference in rFII and hFII. When rFII and hFII were transfected into a warfarin treated transformed human embryonic kidney cell line (293), both proteins were secreted in an under-γ-carboxylated form and intracellular retention was not observed. However, the secretion of rFII was greatly diminished. Cellular retention of under-γ-carboxylated forms is therefore tissue specific, but degradation is not.
A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells
Authors: Ferrer Milagros , Fernandez-Pinel Marta , Gonzalez-Manchon Consuelo , Gonzalez Jose , Ayuso Matilde S , Parrilla Roberto
Published: July 2018

This work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin αIIbβ3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G→A base transition at position 1074 of GPIIb that results in an Arg327→His substitution.

CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of αIIbβ3 complex relative to control cells transfected with wild type GPIIb. Immunopre-cipitation analysis demonstrated that GPIIb synthesis, heterodimeriza-tion, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface.

Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of αIIbβ3. This novel observation of a dominant-negative effect of the mutant His327αIIb subunit provides a molecular basis for the reduced platelet αIIbβ3 content observed in the heterozygous offspring.
Results of a Screening for von Willebrand Disease Type 2N in Patients with Suspected Haemophilia A or von Willebrand Disease Type 1
Authors: Schneppenheim Reinhard , Budde Ulrich , Krey Sonja , Drewke Elke , Bergmann Frauke , Lechler Eberhard , Oldenburg Johannes , Schwaab Rainer
Published: July 2018

A screening program for the detection of patients with von Willebrand disease type 2N (VWD 2N) was carried out in 177 unrelated patients previously diagnosed with haemophilia A and in 199 unrelated patients with VWD type 1 in comparison. By measuring the factor VIII (FVIII) binding capacity of von Willebrand factor (VWF), we detected 13 patients with VWD 2N within 8 unrelated families. The former diagnosis has been haemophilia A in 5 index patients, and VWD in the remaining 3. Included in this study were 14 patients with suspected haemophilia A, whose molecular analysis for mutations in the FVIII gene was unsuccessful. Five of them had VWD 2N. In all patients with the VWD 2N phenotype we were able to identify specific molecular defects in the corresponding DNA sequence of the FVIII binding domain of VWF. The most common defect was R91Q. Four patients from 4 families were homozygous for R91Q, six patients from 3 families were compound heterozygous for R91Q and a silent yet unidentified allele. The VWD 2N phenotype of father, daughter, and son in one family, was based on 2 different genotypes, compound heterozygosity for R91Q and VWD type 1 in the father and the daughter, and homozygosity for R91Q in the son. Two patients from one family were compound heterozygous for T28M and ΔC2680-2685 in exon 18, and one patient was compound heterozygous for a novel candidate missense mutation in exon 18 (E24K) and ΔC2680-2685 in exon 18 which is regarded the most common defect causing severe VWD type 3. FVIII:C values of 0.07 and 0.14 IU/ml were observed in patients with the genotype T28MIΔC2680-2685 whereas in patients being homozygous or compound heterozygous for R91Q, FVIII:C was 0.19 ° 0.071 IU/ml. In contrast to the other genotypes, E24KIΔC2680-2685 is correlated with a more severe haemophilic phenotype with a FVIII residual activity of only 0.01-0.02 IU/ml. This emphasizes the need for inclusion of the factor VIII binding assay in the diagnostic workup of suspected haemophilia A.
Purification Of Factors IX And X From Clinical Concentrate
Authors: Russell G C , Kemble G , Tuddenham E G D
Published: July 2018

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.
Familial And Constitutional Moderate Bleeding Disorder Associated With Defective Platelet Cyclo-Oxygenase
Authors: Lecrubier C , Fouque F , Chignard M , Horellou M H , Conard J , Vargaftig B B , Samama M
Published: July 2018

Three family members from two successive generations showed a moderate bleeding disorder. The bleeding times (Duke and Ivy-Borchgrevink) were both constantly prolonged. Platelet aggregation induced by ADP and adrenaline showed no second wave; collagen at low to moderate concentrations failed to aggregate and release ATP whereas higher amounts aggregated and released. Aggregation and release due to thrombin, ristocetin and synthetic epoxy derivatives (U 44069 and U 46619) were normal. Arachidonate (A.A.) was inactive and was not converted into thromboxane (TX) A2 activity evaluated on the rabbit aorta strip. The patient’s platelets did not respond to A.A. when PRP from an aspirin treated control was added. Platelet phospholipids (PL) were labelled by 14C - A.A. before stimulation by thrombin (T). Radioactivity of the PL of the propositus' platelets was affected with T to a similar extent as 5 laboratory controls, indicating that the phospholipase activity was not impaired. In contrast, no TXB2 was found: 0.9 p. cent as compared to 6 p. cent of total radioactivity in controls.

Our data suggest a deficit in cyclo-oxygenase particularly since the levels of PGE2, PGF2a and PGD2 were within the limits of detection. These results seem to rule out the possibility of a TX-synthetase deficiency with excessive production of anti-aggregating PGs. This study suggests that transmission is autosomal dominant and confirms that cyclo-oxygenase is not needed for aggregation and ATP-release by high amounts of collagen.
Unactivated Plasma Of Children With Cooley’s Anemia Has Serine Protease Activity
Authors: Andrew M , Manno M , Karpatkin M
Published: July 2018

Twelve children with Cooley’s Anemia aged 2-17 had low biologic activity of factors XI, XII and prekaI Iikrein (PK), (mean ± S.E., XI:56%±6; XII:51%±7; PK:59%±6). This did not appear to reflect depressed hepatic function as other factors measured (fibrinogen, I I, V, VI I, VII I, IX and X) were in the normal range. To investigate the possibility that these factors were being activated in vivo and then removed, nonactivated plasma collected in Na Citrate and EACA was incubated with the chromogenic substrate for thrombin, S2238. The substrate was cleaved by every patient tested (34 samples from 12 patients); nm p-nitroanaline (pNA) released/ml/min was 33±5 compared to 3±4 in 20 samples from 10 healthy controls (p <.001). The substrates for plasma kallikrein (S2302) and Xa (S2222) were also cleaved. S2302 showed greatest sensitivity and S2222 the least (mean nm pNA/ml/min; 2302:I85±27; 2238:43±II; 2222:II±4; p <.001). This proteolytic activity was present in fresh plasma and remained stable at -70° for at least I month. It was inhibited by DFP indicating that it was a serine protease. Trasylol, which inhibits plasma kallikrein and plasmin, also inhibited it. It was unaffected by hirudin indicating that it was not thrombin. Children older than 10 years (N=6) had more protease activity than those (N=6) less than 10 (97±23 nm pNA/ml/min vs 24±6; p<.05). Thus patients with Cooley’s Anemia have a serine protease in unactivated plasma which increases with age and may be related to iron overload. We speculate that due to increased tissue iron a zymogen (?PK) is activated to a protease (?kaI Iikrein) which activates XII which in turn activates XI. The activated factors are then removed. This would explain the depletion of XI, XII and PK in these patients.
Anti-Xa Potentiating Effect Of Low Molecular Weight Heparin
Authors: Junker W , Harenberg J , Fussi F , Mattes K , Zimmermann R , Weber E
Published: July 2018

Recently special attention has been drawn to bleeding complications of commercial heparins in patients with increased risk for haemorrhages. Alternative heparin preparations with high antithrombotic and low haemostaseological properties have been developed. We now report on a new low molecular weight (LMW) heaprin (mean MW 5000, 85 USP/mg), which has been obtained by depolymerisation of a heparin from pig intestinal mucosa (mean MW 15000, 154 USP/mg).

In vitro the anti-Xa-activity (chromogenic substrate S2222) was 15% higher for the LMW heparin in a range of 0.01-2.0 USP/ml plasma. No difference was seen on the anti-IIa-activity (thrombin clotting time)and the aPTT for both heparins in the same range. Both Heparins were injected s.c. in a dose of 100, 50 and 25 USP/kg bodyweight into each of six volunteers randomly at weekly intervalIs. The pharmacodynamic effects were controlled for 6-10 hrs by 8-12 blood samples in relation to the dose applied. Increasing dosis the effects of each heparin increased in all test systems. The anti- Xa-activity of LMW heparin was somewhat higher at 100 and 25 USP/kg. At 50 USP/kg the effect of LMW heaprin was in the same range as 100 USP/kg of the original preparation (MW 15000). The factor Ila activity and aPTT were not influenced differently by the two heparins at each dose.

The data indicate, that the LMW heparin presented here may have a more pronounced antithrombotic property by a specific anti-Xa-activity than the compaired commercial heparin. This effect is most pronounced at doses, which have only small haemostaseological effects.
Heparin Concentrations Correlated To The Activated Whole Blood Clotting Time (Act) During Extracorporeal Circulation
Authors: Stenbjerg S , Berg E , Albrechtsen O K
Published: July 2018

Heparin levels and ACT were followed during open heart surgery in lo patients. Heparin was assayed by an amidolytic method using substrate S-2222. ACT was determined with an automated method using celite and glass beads as activators of coagulation. Neither the hemodilution nor the depletion of platelets observed during extracorporeal circulation seemed to influence the ACT. An excellent correlation between the ACT and the actual heparin level was found in each patient with coefficients of correlation ranging from 0.73 – 0.97. A slightly better correlation was noticed for values of ACT below 600 seconds. It was concluded that the ACT is a valuable and reliable tool in control of heparinisation during open heart surgery.
Complement C3 Polymorphism In Greenland Eskimos
Authors: Jørqensen K A , Dyerberq J , Stoffersen E , Nymand G
Published: July 2018

The C3F gene has been associated with atherosclerotic vascular disease. Greenland Eskimos have a very low frequency of acute myocardial infarction (AMI). The C3 polymorphism phenotypes were therefore determined by high-voltage agarose gel electrophoresis in 125 Greenland Eskimos originating from different parts of Greenland and compared with a control group of 1,554 Caucasian Danes. p The C3F gene frequency in the Eskimos (0.056) was significantly lower (p <0.001) than in the control Danes (0.201). These results indicate that genetical factors may also play a role for the low AMI frequency seen in Greenland Eskimos.
Influence Of Blood Flow On Platelet Interaction With The Vessel Wall And Thrombosis
Authors: Goldsmith H L , Karino T
Published: July 2018

Fluid mechanical factors play an important role in platelet aggregation in the circulation and platelet interaction with vessel walls. Theory and experiment show that, at the high rates of shear prevailing in flow near the vessel walls, inwardly directed forces cause single blood cells to migrate toward the axis. At normal hematocrits, however, migration is opposed by an outward dispersion due to the continual, shear-induced interactions between red cells. The latter also leads the less numerous platelets to move in directions normal to the flow and to collide with the wall at rates which increase with the hematocrit. Thus, there exists a mechanism providing for the presence of platelets close to the endothelium and enabling them to adhere to sites of vessel injury.

More important with respect to thrombosis and wall adhesion are the flow patterns and fluid stresses existing at bifurcations, T-junctions, curved segments and stenoses in the arterial circulation, and at valves in the venous circulation. Here, flow is disturbed, the mainstream may separate from the vessel wall and a vortex forms in the region vacated by the flow. Using a glass model of a symmetrical stenosis, we have shown that in the absence of any aggregating agents, platelets circulating and colliding with each other in the orbits of the annular vortex distal to the stenosis form small aggregates which rapidly grow and are trapped in the vortex. Moreover, the adhesion of platelets to the collagen-coated wall exhibit a peak on either side of the reattachment point of the flow. This is explained by curvature of the streamlines carrying platelets to the wall. Detailed studies of vortices having similar flow patterns have been made at bifurcations and T-junctions in models as well as in cleared, fixed arteries, and in veins with bileaflet valves. The phenomena described will be illustrated with a 16 mm ciné film.

Supported by MRC, Canada, and the Quebec Heart Foundation.
Platelet Inhibitory Drugs Improve Collateral Blood Flow After Aortic Thrombosis
Authors: Schaub R , Helenski C , Gates K , Roberts R
Published: July 2018

Complete occlusion of the feline caudal aorta with a thrombus inhibits opening of collateral blood vessels to the hindlimbs. Our previous studies suggest this inhibition results from platelet activation. To test this hypothesis we evaluated the effect of indomethacin (I), aspirin (ASA), and prostacyclin (PGI) on collateral inhibition. Prothrombin times (OSPT), thromboplastin times (PTT), fibrin monomer (FM), and platelet counts were measured in 20 cats before the aortas were occluded by thrombosis. Five cats were untreated. Treatment of the others was started 1 hour prior to thombosis. Five cats received I (20 mg/kg i.v.), 6 cats received ASA (650 mg orally), and 4 cats received an i.v. infusion of PGI (50 ug/kg/hr). Cats were maintained 3 hours post-operatively. Coagulation parameters were measured after the 3 hour period and calculated as a percent of pre-thrombosis values. Collateral circulation was assessed as the time necessary for contrast media to appear in the iliac arteries caudal to the thrombus during aortography. All treated groups were statistically compared to the non-treated group. The OSPT, PTT, and FM values were not significantly changed in any group except for a prolonged PTT in the ASA group. Platelet counts were significantly reduced in non- treated cats (66 ± 4%) compared to treated cats I (96 ± 3%), ASA (97 ± 10), and PGI (97 ± 9%). Contrast media did not appear in the iliac arteries of non-treated cats until 8 seconds injection. In 4 I treated and all ASA treated cats contrast media appeared in the iliac arteries within 2 seconds after injection. The PGI treated cats had an intermediate response. Contrast media appeared in the iliac arteries 4 seconds after injection. These results suggest: (1) Some consequences of arterial thrombosis may be due to humoral inhibition of collateral blood flow. (2) This inhibition is related to platelet activation with release of vasoactive and aggregating agents and not to gross alteration of other clotting factors. (3) Platelet thromboxane A2 maY be the factor promoting collateral inhibition since prostaglandin synthesis inhibitors and antagonists are effective. (4) Inhibition of platelet function prior to thombosis could reduce ischemic damage by enhancing development of collateral blood vessels.