bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation Of DNA Synthesis In Mouse Fibroblasts By Purified Porcine Platelet Secreted Proteins
Authors: Poggi A , Rucinski B , Holt J C , Niewiarowski S
Published: July 2018

Human platelets secrete proteins which stimulate multiplication of fibroblast and smooth muscle cells and appear to be involved in the development of atherosclerosis. We have studied the effect of two purified porcine platelet secreted proteins on DNA synthesis in mouse fibroblasts (3T3 cells). Material released from washed platelets stimulated by thrombin was fractionated with 0.04 M ZnSO4, followed by chromatography on Sephacryl S-200 and heparin-agarose. A number of proteins were eluted from heparin-agarose with a gradient of increasing NaCl concentration including Heparin Binding Protein (HBP) and Platelet Factor 4 (PF4), eluted at 0.7 M and 1.1 M NaCl respectively. Both proteins were homogenous in immunoelectrophoresis and in SDS polyacrylamide gel electrophoresis. Their apparent molecular weights were 6,000 and 14,000 respectively. They showed no proteolytic activity using fibrinogen and D-Phe-Pip-Arg-pNa (S2238) as substrates. At concentrations 500-5000 ng/ml, HBP stimulated 3H-thymidine incorporation into subconfluent 3T3 cells made quiescent by serum deprivation. At the latter concentration the effect was similar to that induced by 3-5% fetal bovine serum. Addition of HBP (5000 ng/ml) also stimulated proliferation of 3T3 cells plated in 1% of fetal bovine serum. In both assays porcine platelet factor 4 was at least 10 times less active than HBP. Mitogenic activities of human platelet basic protein and of human platelet derived growth factor were potentiated by addition of fetal bovine or human serum. By contrast, no synergistic effect was found between these sera or porcine serum and HBP (500 ng/ml) when tested by 3H-thymidine incorporation assay. A possible role of secreted porcine platelet proteins in the development of atherosclerosis in swine is under investigation.
Improvement Of Shortened Platelet Survival Half-Life From The Early To The Late Phase Of Myocardial Infarction
Authors: Chesebro J H , Fuster V , Byrne J
Published: July 2018

The incidence of reinfarction, death and systemic thromboembolism is greatest in the first 2 months after acute myocardial infarction and decreases with time after infarction. Reasons for this are unknown, but increased platelet vascular wall interaction may be a contributing factor. Thirteen patients, mean age 53 years, had a platelet survival determination (platelet half-life calculated by least square analysis) within 1 month (mo) after acute, uncomplicated myocardial infarction (10 transmural, 3 subendocardial); a platelet survival study was repeated 3-8 mo (average 4 mo) later. All patients were on oral anticoagulants and during the time of observation none of the patients had any clinical thromboembolic events.

Ten of the 13 patients had a shortened (<92 hours) platelet survival half-life of 75 hours (avg) within 1 mo after myocardial infarction which increased significantly (P<.01) to 94 hours (avg) four mo (avg) later. Ten control patients with angiographically proven coronary artery disease, stable angina pectoris, and a shortened platelet survival of 85 hours (avg) had a repeat platelet survival 6 mo later that was not significantly changed (mean variability 5%).

The shortened platelet survival that improves with time after myocardial infarction is compatible with the increased incidence of reinfarction, death and thromboembolism early after myocardial infarction. Whether the increased platelet consumption reflects platelet deposition in the coronary vessels or in the left ventricle (as a mural thrombus) is at present under investigation with imaging of III-Indium-labeled platelets.
Antibodies As Probes Of The Conformation Of Fibrinogen
Authors: Plow E F , Edgington T S
Published: July 2018

In order for an antibody to react with a protein, the recognized antigenic determinant must be available at the hydrated surface and be a conformation permissive for antibody binding. The validity of the capacity of antibodies to identify surface-oriented regions has been verified with a number of protein molecules of known three-dimensional structure. Based upon this concept, we have utilized antibodies to defined structural loci of fibrinogen to identify exposed and inaccessible regions of the molecule. The following information has been derived from such studies. I) Regions of the molecule including the C-terminal of the A α chain and the N-terminal of the B β chain, which are accessible to proteases such as plasmin, are surface oriented. The accessibility of protease-sensitive regions to antibodies would be anticipated and serves to validate the approach. 2) The D and E domains are capable of interacting with one another. This interaction qppears to be a native conformational element of fibrinogen and reflects more than the mere abutting of the C-terminal of the E domain with N-terminal of D domain. 3) Fibrin degradation products are immunochemically distinguishable from fibrinogen derivatives suggesting that the fibrin transition results in conformational change. 4) Plasmic degradation is a dynamic process resulting in conformational changes in both the D and the E domains. This suggests either a spatial proximity between a number of structurally distant regions such as the C-terminal of the A α chain and the N-terminal of the γ chain or a remarkable capacity to transmit allosteric effects from one domain to another. 5) Antigenic determinants (μ 10) located within the N-terminal two-thirds of the γ chain are unreactive with antibody suggesting an interiorization or unique conformation which hinders antibody binding. One interpretation of this latter observation is that the γ chain provides a “nucleation center” which is circumscribed by the other Constituent chains of fibrinogen. These results delineate a set of constraints on the conformational format of fibrinogen in solution. With additional antibody probes to specific structural loci within the molecule, it should be possible to establish additional constraints and to further define exposed and interiorized regions of fibrinogen.
Replication Rate Of Endothelial Cell And Internal Surface Of The Aorta During Different Types Of Experimental Hypertension
Authors: Gabbiani G , Chastonay De C , Hüttner I
Published: July 2018

The reaction of the rat aortic intima to hypertension varies in experimental models having different pathogenetic mechanisms. Thus, thickness, permeability to horseradish peroxidase, mitotic index and density of endothelial cells in the aorta are more marked during hypertensive situations associated with high levels of circulating exogenous or endogenous mineralocorticoids than in situations associated with low levels of these hormones. We have correlated mitotic index and cell density of endothelial cells with the internal diameter of the aorta in early and late phases of 3 models of experimental hypertension: 1) aortic ligature between renal arteries; 2) uninephrectony and Na-rich diet; and 3) uninephrectomy, 0.9% NaCl as drinking fluid and administration of desoxycorticosterone acetate (DOCA). Mitotic index and/or cell density were increased compared to controls: l) early after aortic ligature, and 2) early and late after DOCA. The internal diameter of the aorta, measured morphometrically after perfusion with glutaralde- hyde at the mean pressure of each rat, was increased only late after aortic ligature when mitotic index and cell density were normal and late after DOCA when mitotic index was normal and cell density was high compared to controls. These results show that there is no correlation between the internal surface of the aorta and the variations of mitotic index and/or cell density of endothelial cells during different types of hypertension and suggest that during certain types of hypertension mechanisms other than changes in the aortic endothelial surface stimulate the replication of endothelial cells.
Immunological Diagnosis Of Von Willebrand’S Disease (vWD) And Haemophilia A (HAEM A)
Authors: Bloom A L , Davies B L , Furlong R A , Giddings J C , Peake I R
Published: July 2018

Most immunological tests for vWd are based on xenogeneic antisera to factor VIII related antigen (VIIIRAg/WF). Immunoprecipitin methods quantitate the antigen and may indicate abnormal electrophoretic mobility. Several groups have demonstrated polymeric variation of VIIIRAg using radioimmunoprecipitation, large-pore electrophoresis and autoradiography. An immunoradiomeh ic assay (IRMA) correlated with ristocetin cofactor activity in vWd or showed non-parallel dose response curves suggesting abnormal antigen- antibody binding. The IRMA may preferentially quantitate higher oligomers of VIIIRAg/WF. Based on these studies a simplified classification of vWd is suggested. Immunological tests for Haem A originally demonstrated cross-reacting material (CRM) in 10% of patients or in greater number when tested by inhibitor neutralisation (IN) in antibody excess. Several IRMA’s for VIII coagulant antigen (VIIICAg) have been developed. Utilising alloantibodies in a two- site method VIIICAg was quantitated in normal plasma and serum and was reduced in most haemophilic kindreds (CRM- or CRMR). IRMA’s using three different inhibitor igG’s showed slightly different patterns of results suggesting that the antibodies react with different but related epitopes. Only one CRM+ kindred has been detected in over forty tested. The IRMA for VIIICAg has improved discrimination of tests for carriers of Haem A in non-pregnant women and has complemented the VIIIC assay for prenatal diagnosis; results in 48 pregnancies (Feb. 1981) have confirmed the predictive value. Levels of VIIIRAg were normal in 28 patients with combined VC and VIIIC deficiency. Levels of VIIICAg and VCAg (IN) were reduced in many but not all patients. Correlation was noted in (IN) assays using one combination of antibodies suggesting a bio-relationship between the proteins. The results emphasise the role of immunoassays in assessing Vlll-deficiency states.
A Plasma Fibrinogen Fraction With High Sialic Acid Content And Eecngated γ-Chains
Authors: Kuyas C , Haeberli A , Straub P W
Published: July 2018

Having recently been able to separate Bβ- and γ-chains of human fibrinogen heterogeneous with respect to sialic acid, we were faced with the question whether a carbohydrate heterogeneity might be present in the original native fibrinoge.

Using DEAE 52 cellulose and stepwise elution with phosphate buffer human fibrinogen from pooled or single donor plasma could indeed be separated into 3 populations with different sialic acid content. The most acidic fraction contained 30% more sialic acid (8 moles) than the original fibrinogen (6 moles). After reduction the 3 fibrinogen fractions were analyzed by two-dimensional electrophoresis. The first fraction (15%) contained partly degraded Aα-chains and a pattern of Bβ and γ-chains similar to the one described for the main fraction. The latter (70%) consisted of intact Act-chains, two main Bβ- and two main γ-chains and one additional minor band for each chain, all with the previously described carbohydrate heterogeneity.

The third, most acidic fibrinogen fraction (15%) revealed an unusual γ-chain pattern. In addition to the two normal chains, two γ-chains with higher mol. wt. and more acidic IEP were found. The Aα- and Bβ-chains did not differ from those of the original fibrinogen. Again a heterogeneity with respect to sialic acid was visible both in the Bβ- and the normal as wsll as the elongated γ-chains. Desialylation of the elongated γ-chains resulted in only partial normalisation of the IEP.

The binding of EDTA and citrate shows a strong ^-dependence suggesting a (partly) ionogenic binding between charged areas on the fibrinogen molecule and charged groups on EDTA or citrate

It has thus been possible to attribute the recently described elongated γ-chain variants to a particular sialic acid rich subfraction of normal human fibrinogen and therefore to conclude that the observed fibrinogen heterogeneity is at least to a considerable extent of inter- rather than intramolecular nature.
Comparative Effects Of Tissular Urokinase And Urinary Urokinase In The Treatment Of 30 Patients With Pulmonary Embolism And Deep Venous Thrombosis
Authors: Lorient-Roudaut M F , Vergnes C , Bonnet J , Boisseau M R , Bricaud H
Published: July 2018

Two types of Urokinase are actually available for the clinical use : tissular urokinase (tUK), isolated from cell culture and urinary urokinase (uUK), partially purified from human urine. Their clinical and biological effects have to be compared.

In this study 30 patients with acute pulmonary embolism and/or deep venous thrombosis were treated, using tUK or uUK, 4500 units/kg, during 12 hours.

In the clinical field pulmonary angiographies, scintigrams, and phlebographies were obtained before and after the treatment. The biological study was first established on serial determination of fibrinogen, platelets, Quick Time, FDP using two methods and euglo- biiin lysis time. At the end of the treatment plasmatic samples of each patients were studied by immunoprecipitations followed by polyacrylamid gel electrophoresis, in the view of evaluation of the D dimer E complex. Two methods were used (LANE D.A. and al., 1977and WHITAKER A.N. and al, 1980).

The results showed a quick similar improvement of the clinical situation. In return tUK appeared to be more effective on the biological point of view. Significant differences were found in : decrease of fibrinogen, increase of FDP and the presence of D dimer fragments. The better action of tUK, especially on the fibrin depositions, is so far unexplained.
A Monoclonal Antibody To Factor IX
Authors: Goodall A H , Janossy G , Kemble G , O’Brien D P , Rawlings E , Rotblat F , Russell G , Tuddenham E G D
Published: July 2018

We describe a mouse monoclonal antibody with high affinity for a functional site on coagulation factor IX.

Mice were hyperimmunised to factor IX by repeated intraperitoneal injections of between 15 and 20 µg of purified factor IX in complete Freunds adjuvant, over a period of 5 months. Three days after a final boost with 15 /ug of factor IX in saline, the spleen was removed from a mouse that had a serum anti factor IX activity of 200 NIH units measured in the standard coagulation assay. The spleen cells were fused with the HAT sensitive, nonsecreting mouse myeloma cell line, P3-NSI/1-Ag4-1 (donated by C. Milstein) using polyethylene glycol 1500 (40% w/w). After plating at 1 × 106 cells/ml in 2 ml multiwell culture plates (211 cultures) hybrid cells were selected from the mixed cell population by culturing in the presence of HAT medium for a period of 33 days. Hybrid clones of cells appeared from day 10 onwards and culture supernatants were tested for the production of antibody to factor IX in a coagulation assay.

One of the cultures that produced an inhibitory effect in this assay has been subcloned twice and established as a monoclonal cell line that can be maintained in continuous culture or can be grown as an ascites tumour in syngeneic mice. This.cell line secretes an IgG1(k) antibody designated RFF-IX/I, that binds to the coagulation site of factor IX. RFF-IX/I inhibits only factor IX and no other coagulation factor. A solid-phase radiometric assay is also described that detects the presence of RFF-IX/Ia by its ability to bind to factor IX linked to polystyrene beads and that can be used to screen for production of similar antibodies by hybrid cell lines.
Modulation Of Adenylate Cyclase Activity In Platelets By β2-Glycoprotein I
Authors: Schousboe Inger
Published: July 2018

β2GlycoProtein I, purified from human serum, has been shown to bind to negatively charged phospholipids. Such phospholipids axe essential reactants in several surface mediated reactions. In blood coagulation, platelets, to which β2-glycoprotein I recently has been shown to bind, furnish a major portion of these phospholipids. Concomitant with this binding, a change in platelet adenylate cyclase activity has been observed. The purpose of this study is to further investigate the effect of β2-glycoprotein I on the adenylate cyclase activity in platelets.

Human platelets isolated immediately after bleeding were washed in buffered isotonic solutions at low ionic strength. At 0 °C intact or sonicated platelets were incubated with various concentrations of β2-glycoprotein I and prostaglandin E1xs. Intact platelets were then sonicated, platelet membranes isolated and frozen at -180 °C, and adenylate cyclase activity of the membranes determined.

The results showed that adenylate cyclase activity in platelets increases about 4 times when intact platelets are preincubated with β2-glycoprotein I at saturation. This increase is also seen when low concentrations of PGE1 (≤ lµM) are present in the preincubation mixture. There was no increase in adenylate cyclase activity when sonicated platelets were incubated with β2-glycoprotein I.

On the basis of these results it is suggested that in vivo β2-glycoprotein I may have one or both of the following functions, l) It may prevent circulating platelets from aggregation by protecting the negatively charged phospholipids and/or by increasing the adenylate cyclase activity and 2) it may catalyze the deaggregation of aggregated platelets.
Convulxin, An ADP And Thromboxane-Independent Platelet activating Glycoprotein From The Venom Of Crotalus Durissus Cascavella
Authors: Marlas G , Joseph D , Franceschi J P , Lefort J , Chignard M , Fouque F , Vargaftig B B
Published: July 2018

Convulxin(Cx), a high molecular weight glycoprotein which was purified by Sephadex G75 and Sepharose 4B chromatography, aggregates platelets of guinea-pigs, rabbits and humans (thresholds of 20-100 pico M for 400,000 platelets/μl). Aggregation and release reaction are plasma-independent, and do not require DFP-fibrinogen (DFP-fib), which increases the platelet response. Cx is not lytic for platelets. Neither ADP scavengers nor aspirin inhibit 2-4 suprathreshold concentrations of Cx. Bivalent metal chelation and PGI2 antagonize Cx. “Thrombinized” platelets lose granular ADP, and still respond to Cx in absence of DFP- fib. Cx-treated platelets are aggregated by ADP, thrombin (T) and arachidonic acid (AA), but are refactory to Cx and to collagen. Cx triggers release of 14C-AA metabolites from rabbit and human platelets, which was inhibited by phospholipase A2 inhibitors. “T-ized” platelets took 14C-AA and failed to release it if stimulated with T, but did so with Cx. Cxi.v.induces thrombocytopenia in rabbits and guinea-pigs, and bronchoconstriction in the latter, which is not blocked by aspirin. Cx is a very effective platelet-stimulating agent, free from proteolytic, amidolytic, esterasic, phospholipase and clotting activities. It probably interacts with T, ADP and thromboxane-independent receptors, and may share a component or a route with the mechanism triggered by collagen.
Subcutaneous Heparin For Prophylaxis For Recurrent ThromBoembolism
Authors: Caprini J A , Thorpe C J , Torkelson S J , Vagher J P , Reyes A Z Delos , Mitchell J
Published: July 2018

One hundred consecutive patients with thromboembolic disease were treated with subcutaneous heparin to prevent recurrence of deep venous thrombosis (70 patients), pulmonary embolus (21), or both deep venous thrombosis and pulmonary embolus (9). Thrombosis was documented by venography, doppler ultrasound, impedance plethysmography, V-P lung scanning, or pulmonary angiography. The hospitalized patients received intravenous heparin for an average of 12.3 days. Intravenous heparin was overlapped with the first dose of 5000 units of subcutaneous heparin which was then given every 12 hours. Fifteen patients had the subcutaneous dosage increased before discharge and 52 had changes in dosage at some point during therapy according to test results. Subcutaneous heparin therapy averaged 111 days per patient (range - 15 days to 16 months). No episodes of major bleeding occurred, although 5 patients had minor localized eccymosis or rash. Self-injection was well accepted and tolerated by the patients. Clinical examination, hematocrit platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin split products, and thrombelastography were performed every six weeks. Doppler and impedance plethysmography studies were repeated if clinical signs persisted or recurred. Two patients had recurrent nonfatal deep vein thrombosis 3 months after starting subcutaneous heparin therapy. Both of these patients originally had above knee thrombi.

The results suggest that self-administered subcutaneous heparin injections titered to laboratory tests are effective in preventing recurrent thromboembolism without bleeding complications. This approach represents an effective alternative to oral anticoagulant therapy.
A Comparison Of Chr0M0Genic, Flu0R0Genic, And Fibrinogen Substrate Assays For The Determination Of Plasma Heparin
Authors: Bick R L , Arbegast N R
Published: July 2018

Assays for plasma heparin are now widely utilized in the clinical laboratory and have been found to be particularly useful for monitoring heparin levels in patients receiving subcutaneous or intrapulmonary heparin and for assessing heparin reversal following cardiopulmonary bypass surgery. Three heparin assays were simultaneously used for 40 clinical samples obtained from patients receiving subcutaneous or intravenous heparin and from patients,at mid-bypass, undergoing cardiopulmonary bypass surgery. Correlation coefficients (r values) were then obtained. The heparin samples ranged from 0.04 to 5.1 units/ml. plasma heparin. The assays used were (1) chromogenic (Kabi-s2238), (2) Fluorogenic (Dade), and (3) Fibrinogen (Cutter). It was found that all assays agreed well. The r value for Cutter vs. Dade was 0.93, for Dade vs. Kabi was 0.92, and for Cutter vs. Kabi was 0.92. These studies and data would suggest that all three assays are quite acceptable for the determination of plasma heparin levels, and the relative advantages and disadvantages of each relate only to technique, time, and intrumentation requirements. Based upon these studies, ease of operation and automation, the Fluorogenic (protopath) system was chosen for monitoring heparin levels and reversal in cardiopulmonary bypass patients.
Incidence Of Thrombocytopenia During Therapy With Bovine Lung And Porcine Gut Mucosal Heparin Preparations
Authors: Kwaan H C , Kampmeier P A , Gomez H J
Published: July 2018

Thrombocytopenia complicating heparin therapy is always alarming since the bleeding risk is greatly increased. The observations of Bell et al (Ann Int Med. 85:155, 1976) of a 30% incidence of thrombocytopenia in patients receiving bovine lung heparin was not supported by later reports from Powers et al (JAMA 241:2396, 1979) of only 3.4%. However, the latter study involved the use of heparin prepared from porcine gut mucosal. A prospective randomized study of these two respective heparin preparations was carried out at the VA Lakeside Medical Center on 98 patients to whom heparin therapy was given for the treatment of various forms of thromboembolism. All received a heparin dose that prolonged the activated partial thromboplastin time to l1/2-21/2 times the control level for at least 5 days. 86 patients had heparin for 7 or more days. The observation of platelet counts was carried out for 7 days. 10 patients (10.2%) had a fall in their platelet counts to less than 150×l06/dl during therapy with one of the ten having a platelet count of 95×l06/dl on the 4th day of therapy. All but one of the thrombocytopenias were transient, lasting 1-6 days despite continuation of heparin therapy. Of 49 patients who received porcine heparin, 4 (8.2%) had thrombocytopenia while the incidence was 6 out of 49 (12.2%)in those who received bovine heparin. These results suggest that complications of severe thrombocytopenia are uncommon with heparin derived from both sources.
Modified Regimen Of Low Dose Heparin To Improve Prevention Of Venous Thromboembolism (VTE) After Major Surgery
Authors: Cade J F
Published: July 2018

Low dose heparin, widely used in the prophylaxis of VTE, is incompletely effective in some groups of high-risk patients. For example, after major thoracic surgery for cancer, the incidence of scan-detected deep venous thrombosis (DVT) is reduced from 50-60% to 25-30% by heparin in a dose of 5,000 U bid.

The present study compared the use of sodium heparin 7.500 U bid with 5,000 U bid in an attempt to reduce the incidence of DVT further. 71 patients (55 men, 16 women; mean age 61 years, range 40-84) having thoracotomy and resection for cancer were randomized to receive either treatment post-operatively. 125I-fibrinogen leg scanning was performed daily for 10 days.

The incidence of DVT was similar in both treatment groups, being 25% with 5,000 U and 20% with 7,500 U (X2 = 0.25, P = NS). Most bilateral or extending DVT (5/6) occurred after 5,000 U and most transient DVT (3/4) after 7.500 U. The highest incidence of DVT was seen following oesophagogastrectomy (35%). No abnormal bleeding occurred in either group.

Thus, increasing the dose of heparin did not significantly reduce further the incidence of DVT after major thoracic surgery. It is concluded 1) that further modification of low dose heparin regimens, including even individualization of therapy, is unlikely to be substantially more effective in such patients than present standard regimens, and 2) that, since the thrombogenic stimuli are only partly heparin-responsive, they are likely to be multifactorial and therefore more effectively treated with a combined prophylactic regimen (e.g. low dose heparin and blood flow enhancement) than with a single method.
Venous Stasis - Effect On Release Of Plasminogen Activators
Authors: Risberg B , Bylock A , Kjellström T , Stenberg B
Published: July 2018

Venous stasis affects the fibrinolytic system. This is clinically utilized in the stasis test. Regional increase in fibrinolytic activity is found in the blood. By what mechanism the fibrinolytic activators are released is not known. It was the purpose of the present investigation to study the morphological and functional changes in the vessel wall after venous stasis.

Methods: The infrarenal cavai vein in rats was occluded by a ligature. Biopsies were taken at different intervals from 2 to 8 hours after ligation. Morphological changes were studied with scanning electron microscopy. Tissue fibrinolytic activity was examined histochemically.

Results: Stasis was followed by a progressive decrease in plasminogen activator activity in the intimai layer of the vessel wall. At 8 h the decrease was pronounced and the activity significantly lower than control values. The fibrinolytic activity in the adventitial layer was not influenced by stasis. The scanning electron microscopy studies demonstrated a progressive morphological change with destruction of the endothelial cells.

Conclusions: Venous stasis was followed by progressive structural deterioration of the endothelial cells with release of plasminogen activators. Cellular degeneration might be responsible for enhanced release of activators during stasis.
The Density Distribution Of Intact Platelets Following Their Isolation From All Other Blood Constituents
Authors: Shaw T , Martin J F , Chesterman C N , Peninqton D G
Published: July 2018

A method for measuring the buoyant density of platelets which function normally after the procedure, has been developed and critically compared to published methods.

Following velocity sedimentation into a gradient of polyvinyl pyrrol 1idone-coated colloidal silica particles (Percoll) at physiological pH and osmolarity, 90% (n=10, SD=10) of the whole blood platelet population was recovered with leukocyte contamination of .002% (n=10, SD=.001); erythrocyte contamination was less.

After centrifugation to equilibrium through a second continuous linear Percoll gradient, platelet density showed a Gaussian distribution about a mode of 1.0645 g/ml. (n=13, SD=.0015). Leakage of β thromboglobulin (βTG), lactic dehydrogenase (LDH) and serotonin into the gradients was negligible. Intracellular LDH, serotonin and total protein correlated closely with platelet count, but βTG distribution was skewed towards the denser fractions

Platelets recovered from the second gradient showed normal aggregation patterns and produced thromboxane B2 and secreted βTG when stimulated by thrombin or arachidonic acid. Addition of plasma was necessary to produce ristocetin-and ADP-induced aggregation, and significant radioactivity was not associated with platelets isolated from blood to which 125I had been added

Under physiological conditions, arabinogalactan II (Stractan) caused more spontaneous leakage of α-and dense- granule markers than did Percoll. Platelets taken from different interfaces of a discontinuous Stractan gradient were seen to contain platelets of the full density range when they underwent equilibrium centrifugation in linear continuous Stractan or Percoll gradients.

True buoyant density of minimally altered platelets has been measured by this new technique.
In Vitro And In Vivo Studies On CP 1415 S A New Platelet Antiaggregating Compound
Authors: Biaqi G , Niebes P , Roba J , Lambelin G
Published: July 2018

CP 1415 S is a new phenyloxypropanolami ne derivative.

In vitro CP 1415 S inhibits aggregation of human platelets inaucedby collagen (EC50 9 μM) or arachidonic acid (EC50 12μM). It also primary and secondary phases of ADP, thrombin and epinephrine induced platelet aggregation. Effective concentrations are in the same range (10 μM) as concerns the secondary aggregation and higher as concerns the primary phase. In presence of 50 μM CP 1415 S, platelet aggregation is fully prevented. Addition of CP 1415 S to platelets maximally aggregated by ADP causes disaggregation in a dose dependent way (EC50 50 μM); similar effect is observed only with prostacyclin (PGI2) and PGE1

Ex-vivo, the antiaggregating properties of CP 1415 S have been demonstrated after single oral administration to rats and Rhesus monkeys (minimal effective dose 10 and 20 mg/kg respectively). After repeated oral administrations, antiaggregating activity was observed from 3 mg/kg/day in rats.

In vivo, in rats, using the tail transsection model, bleeding time was significantly (p < 0.01) prolonged by 10 mg/kg P.o. CP 1415 S or ticlopidine. CP 1415 S given orally up to 100 mg/kg had no effect on prothrombin time. CP 1415 S at 1 mg/kg p.o., 2 hours before testing, protected rats (p < 0.001) against acute thrombocytopenia induced by i.v. injection of ADP whereas acetyl salicylic acid and dipiridamole were inactive at 100 mg/kg and ticlopidine was unsignificantly active. Prostacyclin-like substance production by rat vascular tissue, measured by platelet aggregation bioassay, was not affected in rats after 5 or 10 mg/kg i.v.

CP 1415 S appears to be a potent inhibitor of platelet aggregation in vitro and ex-vivo. Its ability to disaggregate platelets is currently tested in in vivo models of thrombosis.
Effect Of Prostacyclin (PGI2) On Human Platelet Aggregation And Secretion
Authors: Chesney C M , Pifer D D
Published: July 2018

PGI2,which increases platelet cAMP(Prostaglandins 13: 389,1977),is a potent inhibitor of aggregation and secretion .We stidued the time course of the same return of platelet function after exposure of platelets to PGI2.Sepharose 2B columns were equilibrated with Tyrode’s albumin buffer, pH7.5 (no Ca2+) containing PGI2 (534nM). Platelet rich plasma was applied and eluted with the same buffer. The filtered platelets(GFP) were then subsampled hourly after elution from the column. Fibrinogen was added to finel concentration of 1.7mg/ml. Platelet aggregation(PA) and release of 14C serotonin (5HT),platelet factor 4(PF4), and factor V (FV) were assayed after stimulation of the platelet by collagen(C), ADP,epinephrine(E), arachidonic acid(AA) and ionophore A23187(I). Data representative of 5 separate studies follow.

I(20μg/ml) induced PA was 76%(Ohr),52%(1hr) and 61%(2hr and beyond). Release of 5HT, FV,and PF4 were 60%,1.89u,and 7.97 yg/10 pit, respectively, at time 0 and increased progressively, reaching a plateau at 2 hr. AA(500μg/ml) was 10%(0hr),30%(2hr),68%(3hr) and 8%(4hr). Release of 5HT paralleled PA but release of FV and PF4 remained suppressed for 4 hrs. In contrast α-granule (PF4 and FV)release by C(μg/ml)increased as PA increased while dense granule secretion remained suppressed. PA as well as a and dense granule secretion by ADP (10μM) were minimal during 4 hrs. PA and FV secretion by E (55μM) also remain inhibited for 4 hrs. In spite of this normal dense granule release occurred initially and declined progressively over 4 hours.
The Regulatory Role Of Camp In Induction Of Human Platelet Receptor For Fibrinogen
Authors: Graber S E , Hawiger J
Published: July 2018

Membrane receptor for fibrinogen plays an essential role in adhesion and aggregation of human platelets by allowing fibrinogen to bridge two or more platelets together. Whereas in normal, unstimulated platelets fibrinogen receptor is not available, it becomes mobilized upon stimulation of platelets with thrombin, ADP, and other stimuli. The mechanism(s) regulating availability of membrane receptor for fibrinogen remains unknown. Following our recent demonstration that prostacyclin (PGI2) prevents mobilization of fibrinogen receptor by thrombin and ADP (Nature 1980, 283,195), we investigated the relationship between cAMP levels and fibrinogen receptor availability. Platelets separated from plasma proteins were briefly exposed to a low thrombin concentration (0.05 U/ml) followed by hirudin to inactivate free thrombin. Binding of 125I-fi- brinogen and cAMP levels were determined in parallel samples. A dose-dependent rise in platelet cAMP levels from 3.3 pM to 10.3 pM/108 platelets in response to PGI2 (3×10-9M - 3×108M) was accompanied by a corresponding inhibition of 125I-fibrinogen binding. The degree of the cAMP increment correlated with binding inhibition (r=0.96). The inhibition of 125I-fibrinogen binding by PGI2 was sustained up to 120 min and was paralleled by a persistent rise in cAMP level. Stimulation of platelet cAMP synthesis “from within” by a ribosylation of the nucleotide regulatory component with subunit A1 of cholera toxin also increased cAMP levels and inhibited fibrinogen receptor mobilization.

These results provide evidence that “up and down” regulation of fibrinogen receptor in platelets is linked to changes in cAMP levels induced by different types of adenyl cyclase antagonists and agonists.
Platelet Reactivity In Coronary Heart Disease
Authors: Riddle J M , Lee T G , Rival J , Goldstein S , Stein P D
Published: July 2018

Platelet reactivity was evaluated in 96 patients with coronary heart disease and 72 healthy subjects. Transmission electron microscopy was used in a standardized in vitro method to evaluate these platelets. The degree of surface activation (cytoplasmic spreading by single platelets) and aggregate formation were both recorded. A hyperactive response was defined as >20% of the spread type platelet and/or an increased number of aggregates (>93 aggregates/100 single platelets). Hyperactive platelet populations were found in only 8% (6 of 72) of normal subjects. In contrast, 467> (6 of 13) of patients with stable angina, 45% (10 of 22) of patients with crescendo angina, 67% (8 of 12) of patients with acute subendocardial infarction and 83% (40 of 49) of patients with acute transmural infarction showed hyperactive platelets. The mean percentage of the spread type platelet for the various coronary heart disease groups was 25, 26, 25 and 51 respectively with corresponding mean values of 101, 84, 71 and 123 for platelet aggregates. The reactivity of platelets from patients with coronary heart disease differed significantly from the normal subjects (P < .01). Patients with acute transmural infarction showed a significantly greater platelet reactivity (P < .005) when compared to the other groups of patients with coronary heart disease. Patients with all forms of coronary heart disease exhibited greater platelet reactivity than normal subjects. These data support the concept that increased platelet activity may be an important factor in acute transmural myocardial infarction and perhaps in coronary heart disease in general.
Malondialdehyde Formation Induced By Different Collagens
Authors: Balleisen L , Rauterberg J
Published: July 2018

To get close information about the characteristics of collagen induced MDA formation, platelet aggregation was induced by collagen type I, III a. V in dissolved and fibrillar form and by methylated type I collagen. The MDA formation was measured in the course of aggregation. In case of Meth.c. MDA production is induced also if under certain incubation conditions no aggregation is detectible.

During the pl.aggr. induced by dissolved c. some MDA production was seen before the slope of the aggregation curve increased. Parallel to the increasing slope MDA increased fast, and some further increase over a 30 min. period was observed. For fibrillar and meth.c. MDA formation begins just with the increasing slope and reaches a stable plateau. In case of meth.c. the plateau is reached immediately after the maximum of aggregation occurred, in that of fibrillar c. some min. after maximal aggregation. For meth.c. the results obtained in the incubation system were comparable to that in the aggregation system.

For meth.c. the kinetics of MDA formation in both systems were further defined. In both systems a concentration dependent saturation kinetic was obtained. In the aggregation system the concentration dependent increase of MDA formation was somewhat faster as in the incubation system and reached its saturation level at a lower collagen concentration.

From the increasing and long lasting MDA formation induced by the interstitial collagens we might speculate that in vivo the stimulating activity of the collagens may work for a long time in conditions where the prostacyclin production of the vessel wall is decreased. The characteristics of MDA formation induced by meth.c. suggest that this c. may be a good tool for in vitro studies.
Heparin Resistance In Thrombocytosis And Erythrocytosis
Authors: Wajima T , Maloney T R
Published: July 2018

Heparin is generally considered to be the drug of choice in the treatment of venous thromboembolism. In most patients an adequate anticoagulant effect can be achieved with doses between 20,000 and 30,000 units/24 hrs. Increased heparin requirement or “resistance to heparin treatment” is seen in patients with massive deep vein thrombosis (DVT), pulmonary emboli, severe AT-III deficiency, or some cancer patients.

We encountered heparin resistance in 7 patients, 3 with idiopathic thrombocytosis and 4 with recurrent CVA’s and DVT associated with relative erythrocytosis. In these patients, a therapeutic prolongation of APTT was not achieved by usual doses of heparin, and thromboembolic episodes occurred despite administration of heparin in doses sufficient to prolong APTT to the therapeutic range. The initial PT, PTT, and TT in these patients were normal. Protamine Sulfate test and Ethanol gelation test were negative. Fibrinogen and Factor VIII levels were elevated in all patients.

AT-III levels (by immunodiffusion method) were normal.

Liver and renal function tests were normal. In the patients with idiopathic thrombocytosis, platelet counts were over 1,500 × 109/l. Platelet aggregation by ADP, epinephrine and collagen was normal or slightly increased. Patients with idiopathic thromocytosis were treated with nitrogen mustard and/or busulfan to reduce the platelet count, then treated with heparin.

Patients with relative erythrocytosis had elevated Hct (>55), Hgb (>18 gm%) and whole blood viscosity (>6 with normal <4), but normal red cell mass; plasma volumes were markedly decreased. Platelet counts were normal to slightly increased. Platelet aggregation studies were normal. These patients were treated with hydration and plebotomy to reduce the Hct below 45 and restore normal whole blood viscosity, then with heparin with good clinical results. Correction of thrombocytosis and erythrocytosis is important in optimal anticoagulation.
Hypothesis On The Role Of Platelet Proteins In Atherogenesis
Authors: Farrell R J
Published: July 2018

Two alternative mechanisms are currently proposed for the development of atheromatous plaques:
(i) accumulation of protein and lipid material from plasma as a result of haemorrheological forces (Copley, 1979);
(ii) proliferation of endothelium in response to a platelet-derived growth factor (PDGF) (Ross, Glomset and Harker, 1977).

The former theory implies a continuous process affecting all endothelial cells but varying with anatomical location and other haemorrheological factors. The latter theory proposes the release of PDGF during platelet aggregation and implies an essentially localised process, probably initiated by endothelial injury. Both theories are supported by substantial experimental evidence.

This hypothesis aims to reconcile these two theories by emphasising recently discovered properties of platelet proteins:
(i) platelet factor hi having anti-heparin activity, may favour thrombogenesis by inhibiting physiological analogues of heparin;
(ii) β-thromboglobulin, by inhibiting formation of prostacyclin, may predispose to platelet aggregation and mural thrombus formation;
(iii) PDGF, while normally functioning in the response to intimal injury, may when it is inappropriately present lead to intimal hyperplasia and plaque formation.

It is proposed that these proteins enter endothelial cells, as governed by haemorrheological factors. The functional (rather than structural) changes so induced then predispose to platelet aggregation and thrombosis with furtner aggregation and ultimately to atherogenesis.
Therapeutic Blood Pressure Reduction Related To Coronary Haemostasis And Acute Myocardial Infarction
Authors: Stewart I McD G
Published: July 2018

The purpose of this paper is to summarize some clinical observations upon the relation between therapeutically reduced hypertensive blood pressure and acute myocardial infarction (MI) together with sudden cardiac death (SD), and to invite expert advice upon the manner and extent to which the findings may derive from the effects upon the clotting process of impaired blood-flow through coronary arteries partially obstructed by atheroma, a pathogenic development that, I would suggest, owes little or nothing, at this site, to raised blood pressure.

In a 6-year study on 169 severe hypertensives under treatment the relative risk of infarction in the patients with final diastolic pressures observed before infarction or its avoidance (FDP) in the range <90mm/Hg (Phase IV) was more than five times greater than in patients with FDP in the range 100-109 (p<0.01), the risk starting to rise again in range 110-114, with similar bimodality in the FDP ranges 76-94, 95-109 and 110-140, the ratios of infarction to numbers being respectively 10/41, 4/52 and 15/70..

It is concluded that, in the presence of coronary atheroma hypertension tends to precipitate MI and SD at each extremity of the pressure scale and that hypotensive therapy lowers the incidence of these events at high presure, only for this benefit to be lost if pressure reduction be pressed too far towards “normality”, an imprecise exercise that may from time to time induce severe impairment of coronary perfusion followed by critical myocardial ischemia, arrhythmia and finally MI and SD.

How do the recondite, and to most clinicians mysterious, processes of thrombosis and haemostasis respond.to the situation created by this haemodynamic failure? In what ways may blood viscosity, platelet aggregation, and indeed the clotting-factors themselves, contribute to the potentially fatal clinical outcome? And to what extent may the threat posed by these wayward influences be restrained by modern treatment?
Platelet Deposition In Coronary Vasculature During And After Global Myocardial Ischemia
Authors: Rosenbaum D S , Feinberg H , Levitsky S
Published: July 2018

Recent evidence indicates that myocardial ischemia activates circulating platelets. Open heart surgery imposes global ischemia and platelet activation could lead to intravascular platelet deposition in ischemically injured myocardium. Platelets were labeled with111In and 51cr. The radioactive cells were injected into dogs prior to cardiopulmonary bypass. Following bypass the aorta was clamped to induce 60 min of global normothermic ischemia. The clamp was then removed to reperfuse the myocardium. Tissue biopsy 111In and 51cr activity indexed platelet content and the extent of perfusion respectively. The tissue platelet content in excess of that predicted from the tissue blood content was determined. A substantial increase in platelet content (X = 400%) was found during the reperfusion period following ischemic insult. Although platelets were sequestered in other organs the increases were moderate and unrelated to ischemia. Thus, the significant excess of platelets found in myocardium during coronary reperfusion indicates the presence of platelet thrombi. Low output failure, a complication of open heart surgery, may be the result of platelet microthrombi lodged in the small vasculature.
Effect Of Exercise On Platelet Function In Patients With Intermittent Claudication
Authors: Baker William H , Fareed Jawed , Messmore Harry L , Kniffin Judith , Squillaci Grace , Foldes Marcia
Published: July 2018

Activation of platelets as measured by monitoring thromboxane B2 levels, release specific proteins and quantitation of endogenous platelet aggregates has been reported in patients with angina pectoris. To study the relationship between the platelet-activation and intermittent claudication, 14 patients with demonstrated peripheral atherosclerosis were exercised to tolerance. Segmental limb pressures and platelet function studies were performed both pre and post exercise. A continuous EKG was recorded. All patients exhibited a drop in segmental limb pressures coinciding with complaints of claudication which was consistant with peripheral atherosclerosis. No significant alteration in the platelet release and aggregation pattern was noted in samples obtained prior to and immediately after exercise. Autoaggregation as measured by luminescence (luciferase-coupled) aggregometry to measure spontaneous ADP release and release specific proteins such as 3 thromboglobulin and platelet factor 4 also remained unchanged. Interestingly although 6 of 14 patients showed an abnormal exercise EKG, no angina pectoris was experienced. Although endogenous activation of platelets has been linked with anginal pain, the present study shows that platelet activation as studied by aggregation, release proteins and thromboxane measurement is not associated with intermittant claudication. The reduction in limb blood flow as evidenced by the drop in segmental pressures is most likely due to anatomic pathology rather than platelet mediated rheologic or vasospastic mechanisms.