bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural Analysis of Organelles Produced in Megakaryocytes Cultured from Human Stem Cells
Authors: Breton-Gorius J. , Vainchenker W.
Published: May 2019

Electron microscopic (EM) studies have permitted the elucidation of platelet anatomy while EM cytochemistry has recently revealed four distinct classes of granules. In spite of several EM investigations, knowledge of the production of organelles in developing platelets with in marrow megakaryocytes (MK) is limited. The recent possibility of obtaining human MK colonies in culture which mature with synchrony and derived from a single progenitor present in blood and marrow permits analysis of MK maturation. We determined that platelet peroxidase, demarcation membrane system, microtubules glycogen and numerous granules develop in MK cultured in vitro; a complete maturation proceeding as far as platelet shedding could be observed in some MK derived from marrow stem cells. The origin of the dense granules which represent the amine-storing organelle in platelets is uncertain. We are currently investigating whether the dense granules in cultured MK represent a separate class and attempting to establish the period of their synthesis. We anticipate that the culture method used in conjunction with EM will permit study of pathological megakaryocytopoiesis.
Stability Over Time of the Heterozygous VWD Phenotype
Authors: Miller C.H. , Graham J.B. , Goldin L.R. , Elston R.C.
Published: May 2019

A study has been made of 181 members of two large vWd kindred in the Carolinas. Analysis of the data from first testing showed that the trait, “affected”—meaning abnormal on one or more of 4 tests (BT, VIII:C, VIIIR:Ag, VIIIR:WF)—was transmitted as an autosomal dominant. There were 23, affected members in the smaller (418) kindred, and 57 affected in the larger (750) kindred. Penetrance of the vWd allele was reduced to 58%, and the “affected” phenotype was highly variable, when the tests were scored (+)= normal and (-) = abnormal, 11 of the 16 possible combinations of (+) and (-) were observed. Only 15% of those affected were of the classical phenotype, i.e. abnormal on all 4 tests.

A subsample (22 persons) of the larger kindred was re-tested twice, 1½ and 2 years after first testing. The “affected” phenotype was found to be very stable over time. In a total of 140 additional tests only 18 (12%) shifted from “abnormal” to “normal” or vice-versa; and only 5 of 22 persons (23%) shifted class; 2 shifting from “affected” to “non-affected”, 2 from “non-affected” to “affected” and 1 from “affected” to “non-affected” to “affected”.
Comparison Between Plasma Levels of β-Thromboglobulin and Platelet Factor 4 in Various Diseases
Authors: Matsuda T. , Seki T. , Ogawara M. , Miura R. , Yokouchi M. , Murakami H.
Published: May 2019

Contentrations of β-thromboglobulin (β-TG) and platelet factor 4 (PF-4) in platelet poor plasma were determined using radioimmunoassay in 97 healthy subjects and 307 hospitalized patients. In normal subjects, mean values (and SEM) of β-TG and PF-4 were 37±2 ng/ml and 8.3+1.5 ng/ml, respectively. Levels of β-TG increased with age. A statistically significant correlation between concentrations of β-TG and PF-4 was observed in 30 normal subjects (r=0.77, p<0.001). Levels of these protein increased in cases of DIC, acute deep vein thrombosis, acute myocardial infarction, acute cerebral infarction, transient ischemic attacks, diabetes mellitus with retinopathy or nephrotic syndrome, whereas these decreased in patients with aplastic anemia or acute leukemia. In a case of DIC with suppressed production of platelets in bone marrow, concentrations of β-TG and PF-4 remained within normal limits. There were few cases, in whom discrepancies between levels of β-TG and PF-4 were observed. Following oral administration of dipyridamole (300 mg daily), concentrations of β-TG and PF-4 decreased. From these results, it is concluded that elevation of β-TG ot PF-4 indicates hypercoagulable states.
Effect of High Plasma-Estrogen Level on Blood Coagulation Parameters
Authors: Kim H.C. , Kemmann E. , Shelden R. , Saidi P.
Published: May 2019

Administration of gonadotrophin (human FSH and LH) raises plasma estrogen level, while progesteron level remains unchanged. We studied the effect of high plasma estrogen level on the blood coagulation parameters. Coagulation parameters were measured on seven anovulatory women of ages between 26-33 years, who were undergoing daily gonadotropin therapy to induce an ovulation. Plasma 17-beta estradiol (E2) level increased 5 fold of base-line level during the treatment (fron 114 ± 28 pg/ml to 553 ± 217 pg/ml). Fibrinogen levels increased from 248 ± 38 mg% of base-line to 353 + 78 mg% during the treatment (t=3.17, P < .025). There was a significant positive correlation between E2 and fibrinogen level (r =.762, a=239.99, b=.23). However, there were no significant changes in prothrombin time (P.T.), activated partial thromboplastin time (A.P.T.T.), Factor VIII procoagulant activity (VIII. C), Factor VIII-related antigen (VIIIR:Ag), platelet retention by glass bead columns, or antithrombin III level during gonadotropin therapy. This study indicates that the acute endogenous rise of ovarian estrogen increases fibrinogen level, similar to pregnancy; but it does not have significant effect on the factorVIII-associated activities (FVIII:C & FVIIIR:Ag).
Inhibition of Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation by α1-Acid Glycoprotein
Authors: Andersen P. , Eika C.
Published: May 2019

α1-Acid glycoprotein (α1,-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (0.05 NIH U/ml final conc.), and inhibition was complete with physiological concentrations of α1-acid GP (1.0-1.5 g/1 final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus similar to the effect of heparin previously observed. As opposed to heparin, however, α1-acid GP did not affect spontaneous platelet aggregation. Furthermore, α1-acid GP (in optimal cone.) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus consistent with the previous findings using heparin thrombin clotting time.

Snyder and Coodley (1976) found α1-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α1-acid GP to inhibit collagen-induced platelet aggregation, α1-acid GP may possibly act as an inhibitor of the release reaction though fairly high concentrations (10 mg/ml final cone.) was needed for complete inhibition.
Factors Influencing Thrombin Induced Platelet Release Reaction
Authors: Hattey E. , Binder B.R.
Published: May 2019

To study the effect of pH and platelet counts on thrombin induced platelet release reaction, washed human platelets labeled with 14C-serotonin were suspended in phosphate buffered saline of varying pH values for 15 minutes with thrombin concentrations between 1,1 and 0,068 NIH U/ml of suspension. The amount of serotonin released caused by the thrombin added was dependent on the pH of the incubation medium with an optimum in the range of pH 7,4 -7,6. This effect was more marked with higher thrombin concentrations. The serotonin release controles without thrombin were not influenced by the different pH values and were always less than 10%. The amount of platelets in the reaction mixture influenced the release values not significantly at thrombin concentrations of 1,1 and 0,27 NIH U/ml While with 0,068 NIH U/ml a significant dependence of the release on the number of platelets was observed, resulting in higher release values in platelet mixtures with lower platelet counts.

Therefore it can be concluded that the pH is of major influence on the release reaction especially at high thrombin concentrations while the platelet count is of importance only at low thrombin concentrations.
Prophylaxis of Deep Vein Thrombosis in Gynecology
Authors: Hohl M.K. , Lüscher K. P. , Gruber U. F.
Published: May 2019

There is only scarce data regarding the prevention of DVT in gynecology. Trials using 3x5000 IU/day of Heparin s. c. or 2x2500 IU + 0,5 mg Dihydroergotamine (DHE) s.c./day in gynecological patients have not been published. Therefore we conducted a prospective, randomized trial comparing the two regimens.

Methods: Included were patients at the age of 40 or over undergoing major gynecological surgery lasting more than 30 minutes under general anesthesia. The I125-Fibrinogen-uptake test was used in all patients for the diagnosis of DVT.

Heparin administration: 5000 IU Na-Heparin (Roche) were given s.c. 2 hours before the operation and 8-hourly thereafter for 7 days.

Heparin + DHE administration: 2500 IU Na-Heparin +0,5 mg DHE (Sandoz) were given s. c. 2 hours preop. and 12-hourly thereafter for 7 days.

Results: Patients in all groups were well matched for age, bodyweight, duration of anesthesia and type of operation. 2 (31%) of 64 patients receiving Heparin and 4 (6. 6%) of 61 treated with DHE-Heparin developed positive leg scans (p > 0. 3). Major bleeding, complications, massive intraoperative bleeding and reoperation for postoperative bleeding or wound hematoma were found significantly more often in the Heparin group (10.9%) than in the DHE-Heparin-group (1. 6%) (p < 0. 05). Conclusions: One third of the Heparin dosis is as effective as the full dosis when DHE 18 added. The lowering of the Heparin dosis leads to a lower number of major bleeding complications.
Value of Low Dose Heparin in Prevention of Deep Venous Thrombosis After Acute Myocardial Infarction
Authors: Cade J.F.
Published: May 2019

Low-dose heparin has become widely used in the prophylaxis of venous thromboembolism in surgical patients but its use in medical patients is less well established. In particular, the reported incidence of deep venous thrombosis (DVT) after acute myocardial infarction (AMI) has varied considerably, as has its response to low-dose heparin prophylaxis. In 94 patients admitted with a provisional diagnosis of AMI, treatment was allocated randomly and blindly as either heparin 5,000 U subcutaneously twice daily or placebo. DVT was diagnosed by daily 125-I fibrinogen leg scanning. DVT occurred in 10% of control patients and in 3% of treated patients. All DVT’s occurred in patients with subsequently proven AMI (68% of admissions) in whom the incidence was 19% in those untreated and 5% in those treated. DVT was more common in patients with cardiac failure (14%) than in those without (3%). There was no difference in incidence of DVT between patients with or without clinically significantly arrhythmias or hypotension. Multivariate analysis combining these factors failed to improve the prediction of DVT. It is concluded that patients admitted for acute Coronary Care are a relatively low risk group for DVT. Minidose heparin appears to be warranted only in those with proven AMI and cardiac failure.
The Use of the Fluorogenic Synthetic Substrate Assay for the Clinical Determination of Heparin
Authors: Triplett Douglas A. , Harms Cathy , Huseby Rolf M. , Mitchell Gary A. , Pochron Sharon P.
Published: May 2019

Utilization of a new method (Ref. 1, Mitchell, et. al., Thrombosis Research, Vol. 13, pp. 47-52, 1978) recently described for the determination of heparin has been employed in clinical studies. The method was compared with accepted monitoring techniques such as the activated partial thromboplastin time determination. Subcutaneous, intermittent, and continuous infusion regimens of heparin were evaluated. Comparisons with the APTT results indicated excellent agreement between the two methods with the correlation coefficient of 0.79. Patients on combination of heparin + coumadin were able to be monitored with little or no interference by the synthetic substrate methodology. Continuous monitoring techniques, using the synthetic substrates technology indicated a high debtee of sensitivity and precision for this method.
Correction of the Bleeding Time in a Patient with Hermanskypudlak Syndrome and Mild Von Willebrand Syndrome by Infusion of DDAVP
Authors: Budde U.
Published: May 2019

A patient with a combination of Hermansky-Pudlak syndrome (oculocutaneous albinism and decreased platelet nucleotides) and mild von Willebrand syndrome (F VIII-AHF 0.18 U/ml, F VIII-RAg 0.50 U/ml , vWF 0.29 U/ml) was treated twice with the vasopressin analogue I-deamino-8-D-arginine vasopressin (DDAVP) in a dose of 0.4 pg/kg SW, Sixty minutes after the infusion the factol VIII complex and the bleeding time, which was usually longer than 20 minutes, were normalized, Platelet aggregation and platelet nucleotid content remained unchanged, The correction of the bleeding time lasted long e nough to allow surgery.
Cinemicroscopic Visualization of Shape Change and Motility in Normal and Abnormal Living Human Platelets
Authors: Zacharski L. R. , Allen R. D. , Rosenstein R. , Widirtsky S.
Published: May 2019

Living human blood platelets (P) have been examined with a high extinction differential interference contrast (DIC) microscope capable of detecting structures as small as 20 nm in diameter. During the quarter hour required for complete transformation, the entire shape change sequence is clearly visible, including disk to sphere transformation, extension and retraction of pseudopodia, and spreading and ruffling of the hyalomere. The exocytosis of intact 5-hydroxy tryptamine (serotonin)-containing dense bodies has been observed both by DIC microscopy and by epifluorescence microscopy in P stained with mepacrine. The release of dense bodies is associated with the formation of one or more “craters” in the upper surface of the granulomere. With optical sectioning, it is evident that certain “craters” represent internal chambers of the open canalicular system. Using these techniques, abnormalities in P motility have been observed in hereditary P disorders. In summary, the ability to observe and record permanently on cine film the motility of living P provides a rapid test of P function which allows quantitation of normal vs. abnormal motile behavior.
In Vitro Inhibition of Platelet Aggregatton by the Gelatin Plasma Expander Haemaccel.(R)
Authors: Stibbie I. , van der Plas P.M. , Ong G.L. , de Jong D.S. , Krenning-Douma E. , Comes M. , ten Hoor F. , Nauta J.
Published: May 2019

In a study concerning open-heart surgery we found that, platelet aggregates present in heparinised human blood disappeared immediately after addition of the gelatin plasma expander haemaccel. A study was therefore initiated of the effect of haemaccei on platelet appregation (Payton apprepometer, 4W RPM as controlled by stroboscope, 37°C, final platelet count 200-300 χ 109/1) and compared with the effect of bovine serum albumin (USA) and platelet poor plasma (PPP). Haemaccel powder was kindly supplied by Rehringwerke and contained 0.98% sodium, 0.015% calcium and no measurable potassium. 0.3 ml human platelet rich plasma (PRP) was nixed with 0.2 ml haemaccel (final concentrations 0-20 mg/ml) in Tyrode’s solution (2 mM Ca++. pH 7,4), Haemaccel inhibited apgrepation in both citrated and heparinised PRP induced by collagen, ADP or adrenalin, both in the presence or absence of indomethacin (90/μM), PPP (0.2 ml) and BSA (in Tyrode’s solution, final concentrations 0-16 mp/ml) were also inhihitinp, hut on a weipht hasis less than haemaccel. Different Ca++ concentrations in the Tyrode’s solution did not alter the inhibition by haemaccel. Final pH in aggregation mixtures varied by less than 0.10 for a given experiment. It is concluded that, under the conditions used, haemaccel and USA inhibit platelet appregation, probably by a non-specific mechanism.
Interaction of Factor V and Factor Va with Platelets
Authors: Tracy P.B. , Peterson J. M. , Nesheim M.E. , McDuffie F.C. , Mann K. G.
Published: May 2019

We have used homogeneous single chain bovine factor V to examine the binding of both factor V and factor Va to bovine platelets, as well as to develop a double-antibody radioimmunoassay (RIA) to measure intrinsic platelet factor V. Reaction of the protein with 125I Bolton-Hunter reagent produced a labelled product which retained 90% of its cofactor activity and gave products indistinguishable from native factor V following thrombin activation. When incubated separately with washed bovine platelets, both 125I-factor V and Va underwent saturable and exchangeable binding. There are high affinity binding sites to which 500-900 V(Va) molecules are bound per platelet with an apparent dissociation constant of 3 χ 10-10 M, as well as binding sites of slightly lower affinity (Kd = 3 χ 10-9M) to which as many as 3500 V (Va) molecules are bound per platelet. Thrombin pretreatment of the platelets was not required for the binding of either factor V or Va. The RIA data for Triton X-100 lysed, washed bovine platelets revealed that 400-1000 intrinsic factor V molecules were present per platelet. Factor V clotting assays produced results consistent with the RIA data. These studies suggest that the factor V molecules intrinsic to the platelet are equivalent to the number of high affinity factor V (Va) binding sites present on the platelet memhrane surface. (Supported by Grant HL-17430 and the hayo Foundation).
Observations on Antithrombin-III-Concentration and its Biological Function During Low Dose Heparin Administration in Patients Suffering from Stroke
Authors: Nienhaus Kh. , Wenig Ch. , Wenzel E. , Pfordt L. , Biewer I.
Published: May 2019

A prospective study was performed on 100 patients suffering from stroke (50 patients were treated with 70 USP heparin/kg b.w./12 hours sc., and Simultaneously, the other 50 patients were treated with 0.9% NaCl twice every 24 hours). The concentration of Antithrombin III (A. III conc.) was measured immunologically (Mancini-technique). The “Antithrombin activity” of the patients’ plasma was evaluated photometrically using chromogenic substrate Chromozym TH (incubation of diluted plasma with 3.5 NIH Thrombin, 5, 15 and 30 min., 37°C). In 50 of these patients (N = 100) the A.III conc. as well as the Antithrombin activity of plasma was found to be decreased, compared with the group of healthy blood donors (N = 50). In 30% of the patients A.III conc.was found to be increased while the plasma’s Antithrombin activity was significantly decreased. Normal values of A.III conc. were observed in less than 20% of the patients. A significant higher rate of deep vein thrombosis was observed in patients not receiving heparin treatment (6/45). No correlation was found between A.III conc. or heparin treatment and the severity of cerebral infarction. Also, no significant differences in A.III conc . (Mancinit technigue) were evaluated between these two groups. Significant differences between parents treated with heparin and those not treated were evaluated by the photometrical measurement of Antithrombin activity in the plasma.
The Effect of Alloxan-Induced Diabetes on Tissue Plasminogen Activator Activity of the Rat
Authors: Smokovitis A.A. , Binder B.R.
Published: May 2019

Subcutaneous injection of alloxan (10mg/100g)induced severe diabetes in Sprague-Dawley rats (hyperglycemia, glucosuria, weight loss, polydypsia, polyphagia and polyuria). The effect on tissue plasminogen activator activity (PAA) was studied histocheraically in key organs (heart, kidney, lung, aorta, and caudal vena cava) 4 days, 2, 3, 4, 6, and 8 weeks after induction of diabetes. After an initially increased vascular PAA (release reaction) seen in lung, kidney, aorta, and myocardium, but not in caudal vena cava, a decreased PAA was found in arteries of the renal medulla and particularly of the renal cortex and in arteries of the myocardium two bo eight weeks after the induction of diabetes. Compared to the normal PAA level, the intima of the aorta showed after the initial rise, a fall in two to three weeks, a second rise by four to six weeks, followed by a fall to the normal value at the eighth week. In the lung the initially increased PAA continued to be slightly elevated until the sixth week; by the eighth week it was normal. In the caudal vena cava no changes in the PAA were seen. Of interest are the observed differences in PAA patterns; (1) between arteries and veins, (2) large and small arteries, and (3) arteries in different tissues up to at least eight weeks post induction of diabetes.

Supp-. by the Austrian Acad, of Sciences, Arteriosclerosis research group.
Platelet Hyperfunction in Infants of Mothers with Diabetes Mellitus (DM)
Authors: Stuart M.J. , Elrad H. , Hakanson D.O. , Graeber J.E. , Sunderji S. , Barvinchak M.K.
Published: May 2019

Platelet aggregation and prostaglandin formation was evaluated in 20 control mother-neonate pairs (Grp I), and in 13 pairs where maternal DM was present (Grps II and III). Grp I control mothers demonstrated normal platelet aggregation with their infants showing the physiological impairment in aggregation that occurs in the neonate. Using platelet Malonyldialdehyde (MDA) as an indicator of platelet prostaglandin formation, Grp I mothers and infants demonstrated normal values of 3.20 ± 0.31 (1 SD) and 2.46 ± 0.61 n moles MDA per 109 plts respectively. In the 13 patient pairs, 8/13 diabetic mothers (Grp II) showed platelet hyperaggregability and platelet MDA was increased to 3.92 ±; 0.22. All Grp II infants also manifested platelet hyperfunction and increased MDA formation (p<.005) to 3.37 t 0.67.2/8 Grp II neonates restudied on the fourth day of life no longer demonstrated the hyperfunction present at birth. 5/13 diabetic others (Grp III) showed normal platelet aggregation and MDA formation (3.18 ± 0.17) and their infants showed normal neonatal aggregation and MDA formation (2.27 ± 0.67). In tne acuic with DM platelet hyperfunction and increased prostaglandin formation is present, mese findings appear to be transmitted to the offsprings of such mothers as well. Platelet hpperfunction was not correlated with either neonatal blood sugar or blood viscosity measurements. Platelet hyperfunetion may be the etiologic factor in the increased incidence of both arterial and venous thrombosis that occurs in the infant of the diabetic mother.
Investigation of the Cause and Effect Relationship between Hydrodynamic Forces and the Developement of Platelet Micro - Thrombi
Authors: Kratzer M. , Müller-Mohnssen H.
Published: May 2019

Hydrodynamic experiments using models of coronary artery branchings and PRP show that by drodynamic forces initiate platelet micro-thrombosis under the following conditions: Adhesiveness of platelets and/or of the wall exceeds a minimum value and velocity component perpendicular towards the wall overeares a critical value [v1*), so that platelets interact with the wall. These hydrodynamic conditions appear especially in stagnation point flow. To investigate the particle wall Interaction directly, the pathways of platelets and the velocity field within the stagnation point flaw was recorded down to a wall distance of 10 μm by a laser ultramicrosoope-anemometer. The number of platelets per unit area and time (pd) deposited at the glass wall was measured independently. Results: The number of platelets touching the wall per unit area and time was pt = C χ v1 (C = concentration of species, measured in a wall distance of 0.6 μm), From the values measured the particle wall adhesivity kpw = pd/pt was computed as an empirical value, measured was about ten times lower than the value estimated assuming a linear velocity profile near the wall. The difference of carpetee and measured values suggest that the velocity gradiert near the wall is slower. To prove this assumption the velocity profile was determined by a laser micro-anenniieter down tc a wall distance of 1 um (Kratzer, M. and Reuter, B. Physiology Congress 1979, Kiel). First measurements show a decrease of the velocity gradient near the wall if macroiclecules are present in the fluid.
Prethrombotic State
Authors: Mitchell J.R.A.
Published: May 2019

Blood is the only part of the body which can be repeatedly biopsied. Whether such biopsies will enable a prethrombotic state to be identified depend on three factors:

1. Timing The histology of thrombi sunnests that they often form in a phasic or episodic msnner. Tf any accompanying blood changes are also episodic, detection is difficult, whereas if they are present continuously, the task is easier.

2. The cause of thrombosis If normal blood is instructed to thrombose by a local vesselwall disease, then there will be no detectable blood abnormality, unless the effect of traversing a thrombosing area produces a persistent alteration in blood properties. If aberrant flow patterns produce thrombosis, then again, blood samples withdrawn elsewhere could be normal. If a primary blood abnormality is resoonsible for thrombosis then our chances of detecting this will be better.

3. The tests used A thrombus is rich in platelets, leucocytes and fibrin but we have as yet no satisfactory tests to detect abnormalities since in vitro tests introduce many artefacts (foreign surfaces, anticoagulants etc). If one studies individuals with thrombotic disease, any abnormalities found may be the consequences and not the cause of the occlusion. If one studies fit individuals, then very laroe groups are needed to yield enough thrombotic events for analysis, and comparison with the remainder of the subjects who do not dene lop thrombosis.
Release of ADP from Erythrocytes Under High Shear Stresses in Tube Flow
Authors: Schmid-Schönbein H. , Rohling-Winkel I. , Blasberg P. , Jüngling E. , Wehmeyer A. , Born G.V.R. , Richardson P.
Published: May 2019

ADP stemning from red blood has been shown to “activate platelets” producing shape change, as well as aggregation and release. However, the mode of release of ADP from intact RBC has never been established. Contrary to popular misconceptions, high shear stresses (τ) prevail during natural hemostatic plug formationin arteries and arterioles. Therefore, we tested ADP-release from RBC subjected for 5-100 msec in tube flow (τ-0-200N/m2) during passage through a hollow fiber (0 400 μm, L = 20 cm) with semipermeable walls (AMICON R). Samples from the fluid layer near the wall were ultrafiltered through it and became accessible for chemical analysis. Concentrations of K+, adenosine nucleotides (HPLC), and Hb (In the supernatant) before and after shear exposure were measured. At T > 50 N/m2T K+, adenosine nue Ieotides, and hemoglobin concentrations rose in the supernatant. Only K+ was higher in the u1 trafiltrate than in the latter, whereas total concentration of adenosine nucleotides were not different and hemoglobin did not permeate. There was no difference between the relative molar concentration of total adenosine nucleotides of hemoglobin, i.e. the nucleotides and hemoglobin content of 10-4 and 10-3 of all RBC were liberated. In the ultrafiltrate (ADP) > 2 χ 10-7 M/L, sufficient to activate platelets in the presence of Ca++.
Effect of Calcium and magnesium on Receptor Mediated Binding of Fibrinogen by Platelets
Authors: Marguerie G.A. , Plow E.P. , Edgington T.S.
Published: May 2019

When washed human platelets are stimulated by ADP, specific and saturable binding of fibrinogen is rapidly induced, consistent with a discrete cellular receptor. In the absence of divalent cations (<10-6 M), stimulation of plateles by ADP resulted in a well defined shape change but aggregation occurred only when Ca++ or Mg++ were added at mM levels. Deaggergation occurred only in the presence of Ca++ and apyrase. Binding studies demonstrated that 125I-fibrinogen did not interact with platelets in the absence of divalentations. Both Ca++ and Mg++ supported the ADP induced binding, but approximf~ely half as nany 125I-fibrinogen molecules were bound at optimal Mg++ as opposed to optimal Ca++(1 mM). Ca++ and Mg++ did not produce actitive bipping; and affinity of the platelet receptor for 125I-fibrinogen in the presence of either Ca++ or Mg++ was similar suggesting induction by both divalent ions of a single class of receptor. Under optimal conditions of ADP and Ca++ the maximum number of receptors per platelet differed for individuals with a mean of 49,826 ± 22,321 in 12 normal donors with a range of 17,500 to 82,500. 125I-fibrinogen did not dissociate from the platelet unless apyrase and Ca++ were present, paralleling the aggregation studies. The binding or dissociation of fibrinogen and platelet aggregation or deaggregation are consistent with the hypotpesis that induction of the fibrinogen receptor may be implicated in ADP dependent platelet aggregation. Supported by NIH grant HL-16411.
A Radioimmunoassay for Factor IX (F. IX) Using Staph A
Authors: Lewis R.M. , Reisner H.M. , Lundblad R.L. , Chung K.S. , Roberts H.R.
Published: May 2019

A recently described radioimmunoassay for antibodies to F. IX uses Staphylococcus aureus to specifically precipitate the antibody and boundI labeled F. IX (Lewis et al. Fed. Proc 1979). Tnis method has now been extended to allow for the measurement of F. ix irTETgen by inhibition of the binding of 125I-F. IX to alloantibodies. Purified F. IX as well as F IX in plasma can be measured with high sensitivity (plasma dilutions to 1:2560). This assay was used to characterize hemophilia B plasmas as CRIT, containing normal levels of F. IX antigen, or CRM”, containing severely reduced levels of F. IX antigen. Classification was in agreement with results obtained by inhibitor neutralization using a different alloantiserum. No detectable antigenic differences were observed between normal F. IX, activated normal F. IX (F. IXa) and two abnormal, highly Purified F. IX proteins in this assay. These data indicate the usefulness of the assay for the determination of the antigenic specificity of alloantibodies.
Molecular Biological Aspects of Fibrinolysis
Authors: Collen D.
Published: May 2019

The fibrinolytic enzyme system plays a role in the removal of fibrin from the blood vessels or urinary tract, and also in tissue repair (angiogenesis), cell transformation, macrophage function, ovulation and embryo implantation. Growing endothelial cells, malignant cells, macrophages, granulosa cells and trophoblast cells.produce plasminogen activators which activate plasminogen in the blood or interstitial fluids. Local plasmin formation appears to be a generalized mechanism involved in tissue destruction and repair. Fibrinolysis in the blood seems to be regulated by specific molecular interactions between (tissue) plasminogen activator, plasmin(ogen), fibrin and α2-antiplasmin, the physiological plasmin inhibitor. Plasmin(ogen) contains structures - lysine-binding sites - which mediate its interaction with fibrin and with α2-antiplasmin. When fibrin forms in plasma a small amount of plasminogen is bound via these structures. Plasminogen activator present or released in the blood is strongly adsorbed to the fibrin and activates bound plasminogen in situ. The formed plasmin, which remains transiently complexed to fibrin, both by its lysine-binding site(s) and active center, is only slowly inactivated by α2-antiplasmin, but plasmin which is released from digested fibrin is rapidly and irreversibly neutralized by α2-antiplasmin. Many in vitro and in vivo experiments support this hypothesis for the mechanism of fibrinolysis (Nature 272, 549,1978). Which allows speculation on more efficient therapeutic schemes for thrombolysis.
BAY i 7351, A New Platelet Inhibitor, and antithrombotic Compound
Authors: Seuter F. , Busse W.D. , Hoerlein U. , Boeshagen H. , Hoffmeister F. , Philipp E.
Published: May 2019

4,4′ -Dichloro-N ,N′ -bis [( 1 -methyl-4-piperidinyl) -methyl] -2,2′ di thiobisbenzamide = BAY i 7351 was tested in animal models (rat and rabbit) of traumatically induced thrombosis. After prophylactic administration of small doses of BAY i 7351 (0.3 mg/kg p.o.) to rats the thrombus formation is inhibited (p < 0.05) both in the arterial and the venous system by 79% and 57%, respectively. Further on thrombi already formed (20 and 24 hold) are reduced in weight. The minimal effective doses of these thrombolytic effects in rats are 2 mg/kg p.o. in the carotid artery, and 6 mg/ kg p.o. in the jugular vein, when administered in two single doses of 1 and 3 mg/ kg. The compound is not an anticoagulant or a fibrinolytic drug. It is an inhibitor of platelet aggregation - induced with various aggregating agents including ADP -, in vitro with minimal effective concentrations in the range of 1-10 μg/ml as well as ex vivo (minimal effective dose: 3-10 mg/ kg p.o., rat). Further investigations as to v arious platelet functions, influence on thromboxane and prostacyclin formation will be reported separately. BAY i 7351 is a compound with properties which are considered favourable for treatment of thromboembolic diseases.
The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets
Authors: Gralnick H.R. , Morisato D.K.
Published: May 2019

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.
Abnormal Wound Healing in Experimentally-Induced Factor XIII Deficiency in Rabbits
Authors: Lee Soo Young , Chung S. I.
Published: May 2019

The observation of poor wound healing and abnormal scarring in Factor XIII deficiency reported by Duekert et al., (Throab. Diath. Haenorrh., 5:179, 1960) aroused great interest in the role of this factor in tissue repair. In the present atudy, Factor XIII deficiency was induced in rabbits by intravenous infusion of IgC isolated from goat anti-rabbit platelet Factor XIII, and the deficient state vaa maintained by daily infusion. Control rabbits were given normal goat IgG. After production of standard akin wound the progress of eptthellalizacion, fibroblast proliferation, and collagenous matrix formation was monitored by histology and immunohiscocherais try. In normal rabbits, epithelialization was complete by day 6; fibroblast proliferation by day 10; maximal collagenous matrix accumulation was observed on day 14. In Factor XIII deficient animals, epithelialixation was not completed until day 14; fibroblast proliferation and collagenous matrix accumulation were retarded beyond day 20. These observations suggest that Factor XIII is involved in early phases of wound healing. In an initial attempt to study the mode of Factor XIII in tissue repair, the effect of Factor XIII ou the growth of fibroblast was examined in vitro. However, contrary to an earlier report, the addition of Factor XIII to the culture media shoved no effect on the growth of cells.
Pharmacokinetics of Heparin Administered with and without Dihydroergotamine
Authors: Beermann B. , Lahnborg G.
Published: May 2019

Dihydroergotamine (DHE) enhances the prophylactic effect of heparin on postoperative venous thrombosis. It has been suggested that this enhancement is -caused by interaction with the Phannacokinetics of heparin. The present study was undertaken to evaluate whether DHE influences on heparin kinetics.

Methods Ten healthy volunteers were given iv a) 200 units heparin/kg b) as a + 0.5 mg DHE cl 5000 units heparin d) as c + 0.5 mg DHE. Five subjects were given 0.5 mg DHE only. Venous blood samples were drawn during 8 h after the injections. Concentrations of heparin were detennined using a chromogenic substrate for activated factor X (Coatest Heparin Kabi diagnostica).

Results Area under plasma concentration of heparin time curve in experiments a) and b) averaged 8. 70±1.8l U/ml . h (±SD) and 9. 44±1.04 U/ml . h respectively (NS). The corresponding values in experiments c) and d) were 1.64±0.75 U/ml ; hand 1.7l±0.46 U/ml . respectively (NS). The plasma halflife of heparin in experiment a) was 1.36 h in b) 1.33 h (NS) and in c) 0.64 h and in d) 0.62 h (NS). Injection of DHE caused no increase in heparinlike activity in plasma.

Conclusion It was co ncluded that the beneficial effect of DHE on postoperative thromboembolism is caused by mechanisms other than interaction with heparin kinetics.
Effects of Hypertension and Dietary Saturated Fats on Rat Platelet Functions
Authors: McGregor L. , Renaud S.
Published: May 2019

The purpose of this study was to investigate the effect of spontaneous hypertension on the hyperactivity of blood platelets induced by diets rich in saturated fats. This experiment consisted of feeding 24 weanling male spontaneously hypertensive rats (SHR) (OKAMOTO) and 24 controls (WKY) (KYOTO-Wistar) with diets containing 40% fat for 7 months For 12 rats in each group (SHR or WKY), the diet was rich in polyunsaturated fat (22% corn oil) and for the other 12 rats in saturated fat (38%, particularly in stearic acid) with 2% corn oil to prevent essential fatty acid deficiency. The same experiment was repeated for a period of 5 months. Blood pressure was taken at 3 months (SHR : 194, WKY 150 mm Hg) and 6 months. Blood was removed at 4,5 and 7 months of feeding to determine the clotting time and platelet aggregation to thrombin and ADP. For the same diet, hypertensive rats had a significantly shorter platelet-rich plasma clotting time as compared
to controls (107 sec ± 3.3vs 127 sec ± 2.3, saturated fat diet) (128 sec ± 3.6vs 142 sec ± 2.1, polyunsaturated fat diet). Moreover platelet maximal response of aggregation to thrombin was increased significantly in hypertensive rats (10.9 cm ± 1.6vs 4.8 cm ± 0.8; 8.7 cm ± 1.1vs 2.3 ± 0.4) (Mean ± SE).

In this study, spontaneously hypertensive rats fed purified diets, presented a hypercoagulability and platelet hyperaggregability to thrombin which were markedly increased by the feeding of long chain saturated fatty acids.
Four Sets of Complementary Binding Sites Involved in Fibrin Polymerization.
Authors: Olexa Stephanie A. , Budzynski Andrei Z.
Published: May 2019

Activation of fibrinogen by thrombin releases fibrinopeptides A and B, followed by the formation of an ordered fibrin polymer, probably due to the association of complementary binding sites. The present investigation focused on defining areas of fibrin monomer molecule which are active during polymerization by identifying binding regions on plasmic degradation products of fibrinogen or fibrin. Studies were done in soluble and in solid phase systems. In the first, species were mixed in solution and monitored for complex formation on Tris-glycine gels; in the latter, affinity chromatography on insolublized fibrinogen, fibrin monomer or cross linked fibrin was done. Thrombin-treated fibrinogen, Fragments X, Y, or NDSK bound to Fragment DD in solution as well as to all three insolublized species. Therefore one type of polymerization site is present and available on the Fragment D region of fibrinogen. The complementary site is a thrombin activated region on th e NH2-terminal domain of fibrinogen. Fragments E1 and E2 from crosslinked fibrin complexed with Fragment DD but not with fibrinogen, Fragments X, Y, or D. Fragment E1 and E2 also bound to insolublized crosslinked fibrin but not to insolublized fibrinogen or fiorin monomer. The data indicate that Fragment DD contains a binding site generated in the aligned Fragment D moieties which is inoperative in a single Fragment D. This site is complementary to the fourth site, a thrombin-activated region on the NH2-terminal domain of fibrinogen localized to Fragments E1 and E2. It is concluded that the first two binding sites are active on fibrin monomer molecules but the other two sites become operative only after the formation of fibrin oligomers.
Platelet Glycoproteins in Platelet Aggregation
Authors: Phillips David R. , Jennings Lisa K. , Edwards Harold H.
Published: May 2019

Thrombin stimulation alters the membrane surface of platelets so that specific components on the membrane surface interact. To identify such “aggregation factors”, tne exposed membrane proteins of washed platelets were labeled by lactoperoxidase-catalyzed iodination and tested for their association with cytoskeletal structures. Control, thrombin-stimulated (TS; nM thrombin in mM EDTA to prevent aggregation) and thrombin aggregated (TA; 2 mM Ca++) platelets were treated with 1% Triton X-100. The insoluble material (isolated by centrifugation) from TS platelets, but not unstimulated platelets, had clusters of filamentous material with dense cores about 1 μ in diameter. Each cluster appeared to arise from one platelet and contained proteins with the Mr of actin actin-binding protein and myosin plus a 56K and 90K protein. Triton extraction of TA platelets produced an insoluble material with a similar protein composition as that from TS platelets; however, the filamentous clusters remained- aggregration, indicating tnat membrane components which aggregate platelets were still present. Analysis of iodinated membrane components revealed that all were solubilized by Triton from control and TS platelets while two glycoproteins, termed IIb and III, remained with the filamentous material from TA platelets. This and the observation that platelets lacking IIb and III cannot aggregate [JCI. 60: 535 (1977)], indicate that one or both of these membrane glycoproteins are involved in the direct Interaction of platelets during aggregation.
Structure/Functional Relationships in Serum Lipoproteins
Authors: Gotto Jr A.M.
Published: May 2019

Cumulative data from many areas of biochemistry suggest a universal structural organization for the components of the major classes of plasma lipoproteins.Lipoproteins are macromolecular complexes of protein and lipid assembled such that the polar components (i.e. protein and phospholipid) are found at the aqueous interface in a monomolecular film surrounding a core of neutral lipid (i.e. cholesteryl ester, triglyceride). The lipoproteins seem to transport neutral lipids in the aqueous environment of the circulation and are involved in lipid metabolism. Recently additional functional roles of the plasma lipoproteins have been suggested including inhibition of lymphocyte proliferation and maintenance of immunological hemostasis. From physical and chemical studies of the apolipoproteins and their interaction with phospholipid, a model for the binding of phospholipid has emerged. A unique structural entity, the amphipathic helix, requires the apoprotein to fold into an α-helical conformation with the distribution of hydrophobic and hydrophilic amino acid residues juxtaposed such that the hydrophobic residues interact with phospholipid hydrocarbon regions while the hydrophilic residues interact with the aqueous phase.These structural features can be considered in terms of micel-lar-like properties of lipoproteins. That is, the components of the lipoproteins are in equilibrium with the aqueous phase allowing transfer of components between lipoprotein and with various tissues.
Thrombin Activation of Fibrinogen Polymerization and Crosslinking
Authors: Blombäck B. , Hessel B.
Published: May 2019

The transformation of fibrinogen to fibrin by thrombin in blood occurrs in two, interdependent steps:

where Fbg = fibrinogen; FPA, FPB = fibrinopeptide A and B, respectively. FbI and FbII are two different types of fibrin.

The release of FPA is a prerequisite for release of FPB, which occurrs at an initially slow rate. Release of FPA leads to formation of FbI through end-to-end polymerization (A:a interactions). Release of FPB enables fibrinogen units to bind to the growing polymer by so called “lateral” association (B:b interactions). The initial interaction at the B:b sites leads to enhanced subsequent release of FPB and hence of polymer formation.

The fibrin formed, whether of FbI or FbII type, is rapidly cross-linked with inter γ-chain bonds in presence of Factor XIIIa and Ca2+. Formation of inter α-chain crosslinks is slow in purified fibrinogen - thrombin systems and in platelet poor plasma, but fast in whole blood and in platelet rich plasma indicating a role of the platelets in this process.
Comparison of a New Automated Amidolytic Assay of Thrombin Generation with “ Prothrombin Time” and “Thrombotest”.
Authors: Jonker J.J.C. , van Riel L.H.M. , Paulssen M.M.P.
Published: May 2019

An automated method was developed for the determination of thrombin generation in citrated whole blood during activation by thromboplastin. The thrombin generated was allowed to split the chromogenic substrate Tos-Gly-Pro-Arg-pNA yielding p-Nitroaniline. The comparison with this method and “Prothrombin time” in 50 samples was significant (r = 0,88 P < 0,001) and also in 503 samples with “Thrombotest” (r = 0,73 P < 0,001). This assay may serve as a specific, precise and fast and cheaper alternative for “prothrombin time” and “Thrombotest” in large Thrombosis Services.
The Clinical Significance of Chronic Fibrinaemia
Authors: Haselager E.M. , Vreeken J.
Published: May 2019

From 1970-1978 we investigated the occurrence of circulating fibrin monomers in 3293 patients in the department of general medicine by means of the aethanol gelation test. Positive tests were found in 151 patients (± 5%). In a variety of diseases (sepsis, meningitis, liver cirrhosis, bleeding with shock, malignancy) the test was only positive for a short time (1 or 2 days).

Of all the patients who had a positive aethanol test for more than two days 33% turned out to have a malignant disease. When fibrin monomers could be detected for more than 5 days (chronic fibrinaemia) the percentage of malignancy rose to 75%! Fibrinaemia was highly correlated (97%) with an elevated level of FDP/fdp. In patients with malignancies and elevated levels of FDP/fdp 20% had clinical signs of thromboembolism. However when this was accompanied by circulating fibrin monomers (more than 5 days) this percentage rose to 45%. Only 12% of patients with chronic fibrinaemia had a bleeding tendency.