bluuebunny/crossref_metadata_2025 huggingface dataset:
New Synthetic Peptide Substrates Of -Val-pNA Type For An Insoluble Fibrinolytic Serine Enzyme Extracted From Human Spleen
Authors: Okamoto U , Nagamatsu Y , Horie N , Okada Y , Tsuda Y
Published: July 2018

An insoluble spleen fibrinolytic serine proteinase(SFP) extracted from the human spleen with 2 M NaClO4 and partially purified by the authors was characterized with its enzymatic capability to split fibrin and fibrinogen at neutral pH. Its substrate specificity was much limited; any synthetic peptide substrates for factors of coagulation and fibrinolysis were not hydrolyzed. The present studies were therefore made to search for SFP peptide substrate which mimicked possible natural substrates, using the partially purified enzyme preparation showing single protein band possessing fibrinolytic activity on polyacrylamide gel disk electrophoresis.

Despite of fact that various chromogenic peptide substrates having principal structure of -Arg-pNA and -Lys-pNA and various esters of Lys, Arg, Try, Phe and Tyr were not decomposed by SFP, a clue was however found that Suc-Ala-Ala-Ala-pNA (substrate for elastase) was decomposed by SFP but very slowly. Then, trial to mimic a part of insulin B chain (a substrate for elastase) led us to reach Suc-Tyr-Leu-Val-pNA which was well amidolyzed by SFP with Km of 0.17 mM and Kcat/Km(M) of 22,600. Further, Suc-Ala- Tyr-Leu-Val-pNA was found more appropriate substrate than the former. It was noteworthy that ratio of SFP susceptibility of Suc-Ala-Pro-Val-pNA, Suc-Ala-Pro-Ala-pNA and Suc-Ala-Pro-Leu-pNA was approximately 9:1:0.

Results obtained sofar concluded that synthetic peptide substrates appropriate for SFP are dominantly characterized by the C-terminal-Val; other part amino acid sequences is also significant however.
The Thrombin Catalyzed Activation Of Human Factor V Probed With A Hybridoma Antibody
Authors: Katzmann J A , Nesheim M E , Mann K G
Published: July 2018

The peptides produced during the thrombin (IIa) catalyzed activation of human Factor V (HFV) were investigated using a murine monoclonal antibody. This antibody (HFV-1) binds HFV and activated HFV (HFVa) and releases them at high ionic strength. HFV-1 coupled to sepharose was used to purify the single chain, 330,000 apparent MW procofactor molecule. Gel electrophoresis in SDS indicated that IIa- mediated activation proceeded with the appearance of intermediates with apparent MW of 270K, 205K and 150K and resulted in at least 3 end products with apparent MW of 110K, 93K and 70K. Aliquots of partially and fully activated HFV were chromatographed on the HFV-1 affinity resin. The peptides not associated with HFVa were obtained by washing the column with 20 mM imidazole, 0.154 M NaCl, 5 mM CaCl2, pH 6.5; the peptides noncovalently bound to the epitope-containing peptides were obtained by elution in the above buffer made 5 mM EDTA in place of Ca++ and the epitope-containing peptides were eluted in buffer made 1.2 M in NaCl. The 150K intermediate was released in EDTA and the 205K and 270K peptides were eluted in high salt. Of the end products of activaion, the 110K peptide was not retained by the coliman, the 93K peptide was released in EDTA, and the 70K peptide was released only at high ionic strength. These data indicate that the 270K and 205K intermediates, and the 70K end product share the antigenic site involved in the interaction with the monoclonal antibody. In addition a Ca++- dependent interaction between the epitope-containing domain and the 150K intermediate and 93K end product is suggested. From this data and by analogy with the bovine protein, we suggest that HFVa is a Ca++-dependent multiple subunit protein consisting minimally of two peptides of apparent MW of 93,000 and 70,000.
Activation Of Human Factor VII By Activated Factors IX And X In Purified Systems
Authors: Masys D R , Bajaj S P , Rapaport S I
Published: July 2018

Factor VII activity, as measured in a one-stage clotting assay, increases when whole blood is clotted in glass. Prior studies in this laboratory using factor-deficient plasmas suggested that this factor VII activation was due to activated factor IX (IXa). We therefore studied activation of VII by IXa and by activated factor X (Xa) in purified systems. Human factors II, VII, IX, and X were each purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis. Reaction mixtures of VII, IXa or Xa, and other cofactors and enzymes were made, and subsampled for VII activity. The activation state of VII was judged by comparison of one-stage clotting assay to a coupled amidolytic assay using a synthetic substrate. In the presence of phospholipid (PL) and calcium (Ca), both IXa and Xa activated VII 25 fold; however, Xa was roughly 800 times more efficient than IXa. In the absence of PL, Xa was roughly 20 times more efficient than IXa, in Ca-containing solutions. Only slight activation of VII by either enzyme occurred in the absence of Ca. The addition of prothrombin (II) markedly slowed activation of VII by both Xa and IXa; however, this effect did not occur if fully-decarboxylated II was used. The addition of anti thrombin III and thrombin-modified factor VIII at physiologic concentrations did not change rates of VII activation by IXa or Xa.

These results confirm the ability of IXa and Xa to activate factor VII at physiologic concentrations in purified systems. The higher relative efficiency of Xa over IXa under all conditions studied contrasts strikingly with observations in whole plasma systems where the VII activation measurable after clotting is greater in X-deficient than in IX-deficient plasma. The activation of VII by Xa and IXa may serve as an amplification loop in the generation of clotting by either “intrinsic” or “extrinsic” cascades.
Antiplatelet Agents In Diabetic Lower Extremity Vascular Disease. A Va Cooperative Study
Authors: Colwell J
Published: July 2018

Peripheral vascular disease is a devastating complication of diabetes mellitus. Post-operative vascular death rates are 10% or more following amputation for gangrene in diabetic patients, and three year mortality approaches 30%. An additional 20-30% of patients require subsequent amputation for diabetic gangrene. In view of these statistics, and because of the postulated role of the blood platelet in diabetic vascular disease, a collaborative study on antiplatelet agents was begun in 10 VA medical centers in 1977. In a double-blinded study, aspirin (325 mg tid) plus dipyridamole (75 mg tid) or placebos are given to adult diabetic males who had suffered a recent amputation for diabetic lower extremity vascular disease. End points are major vascular events after at least 3 years of follow-up.

Recruitment of 231 subjects was completed by May, 1980. Baseline characteristics are well matched in both groups. Mean age is 59.6 years, duration of diabetes 12.7 years, smoking history 33.3 pack years, and treatment with insulin 68%. Previous myocardial infarction, congestive heart failure, and/or cerebrovascular disease is present in 17-18% and retinopathy is present in 40% at entry. About 43% have more than one vascular complication at baseline.

Mean duration of follow-up is now 18 months. Major vascular events in separate patients are: 40 amputations, 31 deaths, 5 myocardial infarctions. Numerous less serious vascular events have also occurred, as have multiple events in single patients. Therapy has been stopped in 17% of patients, but rarely due to drug side effects. Compliance with therapy has been good.

We conclude that this study will provide important new information on the natural history of lower extremity vascular disease in diabetes. It will also provide definitive data about the efficacy of antiplatelet agents in diabetic vascular disease.
Prevention Of Fatal Postoperative Pulmonary Embolism With Heparin-Dhe Or Dextran
Authors: Gruber U , Gorgerat J , Torhorst J
Published: July 2018

Previous trials studying prevention of postoperative fatal pulmonary embolism (p.e.) concern elective general surgery. We have terminated a prospective, randomized multicentre trial comparing the effectiveness of s.c. heparindihydroergotamin (DHE) versus i.v. dextran in surgery of the musculoskeletal system including both emergency and elective cases. 14 Swiss hospitals participated. All adults operated upon were included and followed up for 6 weeks. 5000 IU heparin together with 0.5 mg DHE were given preoperatively and 12-hourly thereafter for at least one week. Patients in the dextran group received 6% dextran 70 in saline, a total of 3 × 500 ml. 8001 patients were admitted to the trial and 7413 fulfilled the criteria of the study. 5963 patients had been operated electively, 1450 as an emergency. 4368 patients had general anesthesia, 2259 were operated upon under epidural and 797 had other anesthetic procedures. 55 patients died during the first 6 postoperative weeks, the autopsy rate being 69%. 9 patients in the dextran group died of p.e. In 6 cases the pathologist decided the pulmonary embolus as being the only cause of death, in 3 the embolus was considered a contributory cause of death. In the heparin-DHE group 6 patients died of p.e. In 4 cases the embolus was the sole cause of death, in 2 it was contributory. These differences in the incidence of p.e. are statistically not significant. There were however significantly more bleeding and allergic complications in the dextran than in the heparin-DHE group.

Conclusion: Both s.c. heparin-DHE and i.v. dextran are equally effective for the prevention of fatal pulmonary embolism. Heparin-DHE causes less bleeding complications.
Thrombin-Induced Responses In Human Platelets Differ In Their Requirement For Receptor Occupancy; Evidence For Tight Coupling Between Occupancy And Formation Of Phos- Phatidic Acid
Authors: Holmsen Holm , Dangelmaier Carol A , Holmsen Holm-Kjetil
Published: July 2018

Thrombin causes secretion of dense granule constituents from platelets. The time course and extent of this response is unaltered if thrombin is rapidly neutralized after secretion has started, indicating that a sustained occupancy of the thrombin receptors is not required.

Using a 50-fold excess of hirudin to neutralize thrombin, we show here that the shape change and aggregation responses also are independent on sustained receptor occupancy. In contrast, the secretion of acid hydrolases, liberation of [3H-]arachidonate (from phospholipids in platelets prelabeled with [3H]arachidonate) and formation of [32P]phosphatidic acid (in platelets prelabeled with 32P-orthophosphate) induced by thrombin were immedately stopped by hirudin. However, the incorporation of 32P into phosphatidylinositol and breakdown of [3H]phospha- tidyl inositol continued unaffected after thrombin removal. Thrombin-induced platelet responses can thus be subdivided into two classes according to their requirement for receptor occupancy: The first class (shape change, aggregation, dense granule secretion phosphatidylinositol breakdown and phosphatidylinositol synthesis) requires a short, initial occupancy, while the second class (acid hydrolase secretion, arachidonate liberation and phospha- tidic acid synthesis) requires an occupancy that is sustained as long as the response is executed. The increased turnover of phosphoinositides is a cyclic process, and our results show that only one step - the formation of phosphatidic acid from diacylglyceride and ATP - is tightly coupled to receptor occupancy by the agonist.
Adrenaline Activates Human Platelets But Does Not Cause Primary Aggregation If Thrombin Generation Is Inhibited By Hirudin
Authors: Cazenave J P , Sutter A , Hemmendinger S , Wiesel M L , Lanza F , Daver J
Published: July 2018

Adrenaline(ADR) affects human platelets(PLAT) by binding to a receptors,inhibiting adenylate cyclase and translocating Ca2+ across the plasma membrane. In citrated PLAT-rich plasma(CIT-PRP), ADR induces primary aggregation(1stAGG), which may be followed by 2ndAGG due to the activation of the arachidonate(AA) pathway and the release(REL) of ADP. ADR acts synergistically with other aggregating agents. In contrast, ADR does not aggregate suspensions of washed human PLAT(SWHP) and hirudin(HIR)-PRP. To determine the role of traces of thrombin(THR) on AGG and REL of 14C-5HT of prelabeled PLAT induced by ADR,we have used SWHP and PRP anticoagulated with CIT(13mM), HIR(30 U./ml) or both and examined the effects of addition of HIR(30 U./ml),a specific inhibitor of THR. ADR(1-10μM) does not cause AGG or REL of SWHP,even in the presence of added HIR or CIT. However, ADR(1-10μM) potentiates the effects of ADP and AA on AGG and REL. This is not inhibited by HIR. THR alone (0.02 U./ml) causes shape change of SWHP,addition of ADR(4.5μM) causes extensive AGG and REL, which are inhibited by HIR.In CIT-PRP, ADR(1-10μM)causes 1st AGG followed by 2ndAGG and REL, addition of HIR to CIT-PRP has no effect on AGG and REL. Aspirin(ASA) and/or CP/CPK inhibit 2ndAGG-induced by ADR in CIT-PRP. Addition of ASA+CP/CPK+HIR does not inhibit ADR-induced 1stAGG in CIT-PRP. After addition of CIT to HIR-PRP, ADR does not AGG PLAT. In contrast, ADP causes 1stAGG without REL in HIR-PRP and addition of CIT to HIR-PRP induces 2ndAGG and REL. However, ADR causes 1stAGG and REL, if PRP was prepared from blood to which HIR has been added at the same time of CIT or later. In conclusion: 1) ADR does not cause 1stAGG in SWHP or HIR-PRP if the PLAT have not been exposed to THR during their isolation; 2) traces of THR change the response of PLAT to ADR independently of the effect of CIT on Ca2+ concentrations; 3) the use of CIT-PRP does not prevent completely THR generation.
The Endogenous Lectin Of Human Platelets Is An α-Granule Component
Authors: Gartner T Kent , Gerrard Jonathan M , White James G , Williams Danny C
Published: July 2018

Thrombin-activated platelets express a hemagglutination activity (enhanced lectin activity) not expressed by nonactivated platelets and this agglutinin mediates the direct platelet-platelet interactions which cause the aggregation of thrombin-activated platelets. The enhanced agglutinin activity of thrombin-activated platelets is composed of two components: a plasma membrane-bound component and a soluble component. The expression of both forms of the agglutinin activity is dependent upon secretion. The purpose of this study is to characterize the platelet-bound and platelet-free (soluble) agglutinin activities of activated washed platelets from patients with the bleeding disorders, congenital afibrinogenemia, Glanzmann’s thrombasthenia, gray platelet syndrome, and Hermansky-Pudlak syndrome. Thrombin and A23187 activated afibrinogenemie, Hermansky-Pudlak and thrombasthenie platelets had normal platelet-bound hemagglutination activity. Gray platelets activated by the same agents had deficient platelet-bound hemagglutination activity. In contrast, thrombin-activated afibrinogenemie, gray and thrombasthenie platelets lacked platelet-free hemagglutination activity. Only thrombin-activated Hermansky-Pudlak platelets had a normal level of platelet-free hemagglutination activity. On the basis of these results and the distinguishing characteristics of the defective platelets, it is concluded that the α-granules are the origin of the enhanced hemagglutination activity. Furthermore, it is suggested that the insufficiency of the platelet-bound agglutinin may be the cause of the inability of gray platelets to aggregate normally in response to thrombin.
Platelet Factor 4 Binding Proteins
Authors: Walz Daniel A
Published: July 2018

Platelet factor 4 (PF-4) is a platelet alpha granule specific protein with a high affinity for heparin, and a progressively decreasing affinity for other glycosaminogly- cans. PF-4 is reportedly bound to a proteoglycan (PG) carrier within the platelet and can be dissociated by either high salts or by the presence of heparin. In an effort to isolate and characterize this PG, we synthesized PF-4 resin by carbodiimide coupling human PF-4 to diami nobutyl -Sepharose. Platelet lysate, which had been previously passed over heparin-Sepharose, was applied to the PF-4 resin. PG was determined by the carbazole reaction. All of the carbazole reactive material applied to such columns bound, and was subsequently eluted with 1.0 M NaCl. Gel filtration of this fraction on Sepharose 6B resolved 3 distinct components, the largest of which was exclusively carbazole positive. The product was found to contain: 13% protein; 35% hexuronic acid; 17% neutral sugar; 35% amino sugar, identified as galactosamine. This PG is similar to the chondroitin sulfates. PG antisera, when tested against sucrose gradient fractionated platelet homogenates, localize this material in the alpha granule. This PG does not bind 8-thromboglobulin proteins. PG-PF-4 complexes, as judged by filtration data, can be readily dissociated by heparins. This PG is not an exclusive PF-4 binding protein. A 70,000 dal ton protein has also been isolated from the piatelet lysate. Upon reapplication to PF-4 resins, it quantitatively binds and is dissociated only by molar salts. This 70,000 dal ton is not albumin and appears to be released upon platelet activation. It is unresolved as to the ability of this 70,000 protein to compete with PG for PF-4 binding affinity. The concentration (yield) of the 70,000 protein is greater than that of the PG. Studies are currently underway to further characterize this new protein and determine its effect on PF-4 release, quantitation by RIA and plasma half-life.
Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein
Authors: Schmaier A H , Kuchibhotla J , Colman R W
Published: July 2018

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.
Reduced Invaginated Platelet Membrane In Idiopathic Thrombocytopenic Purpura
Authors: Nouvel C , El-Khoury M , Liverato C , Sié A , Robert A , Boneu B
Published: July 2018

An indirect approach of the amount of invaginated platelet membrane has been provided by size measurements of platelets submitted to an hypotonic shock. We investigated this new morphological parameter in 15 normal subjects and in 11 ITP.

Platelet volume was measured by the electronic method (Coulter counter, channelyser C 1000) on EDTA-PRP and on EDTA-PRP diluted with distilled water. One min. after dilution, paraformaldehyde (final concentration 0.08%) was added to prevent any return to the initial volume by an active process. Platelet lysis was quantified by LDH assay. Isoton was diluted in the same proportion. Arithmetic mean platelet volume (MPV) was calculated ; graphic analysis of the data allowed to determine the mode and the standard deviation (SD) of the log volume distribution.

An inverse linear relationship between MPV and the log of osmolarity was observed. The maximum MPV increase without any detectable platelet lysis was obtained at 120 m Osm. For lower osmolarities a progressive lysis occured without further volume increase.

Macrothrombocytosis and increased SD of the log volume distribution were found in undiluted ITP-PRP (12.2 fl and 0.31 respectively versus 9.2 fl and 0.27 in controls). After hypotonic treatment, the MPV increased in ITP and controls (respectively 17.2 fl and 15.9 fl) but the relative increase was lower in ITP than in controls (42% and 63% respectively) ; the SD of the log volume distribution was unmodi fied.

These results suggest that large platelets occuring in ITP possess a reduced amount of invaginated plasma membrane.
The Effect Of Serratia Marcescens Protease On The Aggregation Of Human Platelets By Von Willebrand Factor And Thrombin
Authors: Cooper H , Bennett W , White G , Wagner R
Published: July 2018

Serratia marcescens produces an extracellular metallo-protease (SP) that selectively attacks the surface of human fixed washed platelets and renders them unresponsive to human or bovine von Willebrand factor (vWF). SP treated fresh washed human platelets were studied for [14C]serotonin release, alterations in surface glycoproteins, and aggregation by bovine vWF and thrombin. Platelet membrane glycoproteins were identified by conventional discontinuous gel electrophoresis techniques using periodic acid Schiff reagent (PAS) to stain for carbohydrate. With SP concentrations above 0.6 μg/ml, there was loss of a single PAS band of ~180,000 MW, corresponding to GPlb. With an even more sensitive technique employing [3H]labelled platelets and autoradiography no significant loss of any of the other membrane glycoproteins was found, even at SP concentrations of 20 μg/ml. Supernatants from these treated platelets showed only a single glycoprotein of ~125,000 MW. The use of SP alone with fresh platelets did not cause aggregation or release. SP treatment did cause a complete loss of both serotonin release and aggregation by bovine vWF, but had only a minimal effect on the release and aggregation caused by thrombin. These studies show that SP selectively cleaves GPlb from the platelet surface. The cleaved fragment is smaller than glycocalicin (145,000 MW), suggesting that SP cleaves at a different site than the Ca2+ dependent protease(s) normally present on the platelet surface. These studies suggest that the 125,000 MW fragment cleaved from GPlb is essential for vWF to cause normal platelet release and aggregation. On the other hand, it appears that cleavage of the majority of GPlb is of only minor importance in the release and aggregation mechanism mediated by thrombin.
An In Vivo Model To Assess Biomaterial Thromboresistance
Authors: Rodvien R , Robinson J , Mitchell R R , Litwak P
Published: July 2018

A minimally invasive model has been developed in which control and test No.5 French catheters are passed retrograde from the lingual to the carotid arteries. Goats are given autologous lll In platelets 48 hours before catheter placement, and, after placement, scanned externally and continuously for 3 hours. Net radionuclide retention occurs for the first 30 to 90 minutes after which lll ln leaves the catheter. Simultaneously placed polyethylene (PE) catheters are non-interactive; the weight of thrombus recovered/cm and platelets/cm for 18 goats (36 catheters) is 19.8 mg ± 9.8 and 8.87 × 108 ± 7.5 respectively. Thrombus is evenly distributed along the axial length of the PE catheter, increasing effective catheter diameter by 44%. Correlation between recovered thrombus/cm catheter and net platelet retention/cm catheter is 0.924 for PE. Various catheters exposed to 0.2 mg/ml albumin or 4 mg/ml albumin retained albumin but accumulated equal amounts of thrombus and platelets as untreated PE catheters. Glutaraldehyde used on albuminated catheters also did not change thrombus and platelet retention. Less thrombus was recovered and less platelets retained by PVC, BiomerR and teflon than by PE catheters. This model provides an excellent method to evaluate different polymers fabricated as catheters, both to understand thrombus growth and dissolution, and to choose among catheters for clinical use.
The Fate Of Surface Bound Heparin And The Long Term Thromboresistance Of Heparinized Materials
Authors: Goosen M F A , Sefton M V , Hatton M W C
Published: July 2018

Displacement by plasma of radiolabeled thrombin and radiolabeled thrombin-antithrombin III inactive complex from a heparinized surface (heparin-PVA) was measured and found to be significant. For example, 63% of the thrombin and 90% of the complex that could not be removed by PBS alone was displaced by heat defibrinated plasma. Preliminary characterization (molecular weight, antithrombin III content) suggests that the eluting product consists largely of thrombin-antithrombin III complex and post complex antithrombin III. Heparin polyvinyl alcohol (PVA) gel beads were prepared by acetal coupling of the heparin to PVA using glutaraldehyde with MgCl2 catalysis. Although permanently bound to the PVA (heparin removal rate was 1.67 × 10-2 mg/g gel⋅min), the heparin retained at least part of its activity in thrombin time, recalcification time, chromogenic substrate and AV shunt assays. Thus, heparin need not be lost from a surface to impart thromboresistance. Our results further suggest that the resulting surface bound thrombin-antithrombin III complex can be displaced from the surface by a component or components of plasma (antithrombin III?) indicating that the surface bound heparin does not become saturated with inactive complex. These results support the argument that heparinization can be an important means of preparing the materials needed for the development of improved cardio-circulatory assist devices and blood handling procedures.
The Duration Of Thrombotic Activity In Maturing Prosthetic Vascular Grafts In Man
Authors: Goldman M , Simpson D , Hawker R J , Norcott H , Drolc Z , McCollum C N
Published: July 2018

Prosthetic graft occlusion is most frequent in the early postoperative period when the luminal surface is highly thrombogenic. It is generally believed that graft maturation ultimately results in a non-thrombogenic surface. The accumulation of 111-Indium labelled autologous platelets in Dacron aorto-femoral grafts has been measured one week following surgery and at intervals of 3 months to 1 year.

Platelets from 9 patients were labelled with 111-In-oxine and reinjected. Isotope emissions over the graft and a reference site (aortic arch) were measured daily for 7 days and gamma camera images taken on alternate days. Graft thrombo-genicity was calculated as the daily rise in the ratio of emissions graft/reference.

All grafts, regardless of age, accumulated platelets and were imaged by gamma camera. Mean thrombogenicity (± SEM) one week after surgery was 0.207 ± 0.037 compared with 0.08 ± 0.025 at follow-up (p <0.01). The platelet survival during the early study was reduced at 6.805 ± 0.61 days but had recovered to a near normal value of 8.57 ± 0.78 days (p < 0.001) at follow up.

Although at a reduced rate, platelet accumulation still occurs on Dacron grafts 1 year following surgery despite the presence of normal platelet survival times.
Comparative Thrombogenicity And Healing Of Vascular Prostheses
Authors: Clagett G P , Graeber G M , Hufnagel H , Carter R , Gregory W , Robinowitz M , Langloss J M , Maddox Y , Ramwell P
Published: July 2018

Thrombogenicity and healing characteristics were evaluated for 3 commonly used vascular prostheses: expanded poly-tetrafluorethylene (PTFE), knitted Dacron (KD), and velour knitted Dacron (VKD). Adult mongrel dogs were randomly allocated to receive thoracoabdominal aortic prostheses (6 animals for each type prosthesis) according to a randomized block design. Prostheses were 28-32 cm in length and 10 mm in diameter. Platelet survival (PS) studies were performed pre-op and every 6-8 weeks post-op for 1 year. PS data were subjected to analysis of variance and are shown {X ± SEM} below.

PS, normal in each group pre-op, was shortened by all prostheses post-op; however, VKD reduced PS significantly more than KD and PTFE. At the end of 1 year, PS in animals with KD returned to normal but remained shortened in those with VKD and PTFE. Animals with VKD had the shortest PS throughout. Prostheses were then removed to assess coverage of luminal surface with prostacyclin-producing pseudointima. KD demonstrated the most complete coverage (90.6±5.5% of surface area) followed by VKD (56.3±9.6%) and PTFE (34.0±5.3%), p<0.02. These studies demonstrate that important differences exist among vascular prosthetic materials in initial reactivity with platelets following implantation and in subsequent rate and degree of healing.
Structure Abnormality Of Fibrinogen Metz And Its Relationship To The Clotting Defect
Authors: Henschen A , Southan C , Soria J , Soria C , Samama M
Published: July 2018

Fibrinogen Metz was discovered in a young woman and was associated with a mild bleeding disorder and repeated abortions. Her parents are cousins and both have an abnormal fibrin formation but less intense than in the propositus.

Fibrinogen Metz is therefore an homozygous case characterized by an unclottability of citrated sample, even in the presence of calcium and large quantities of thrombin. In addition, the clotting time is prolonged when the blood is collected in the absence of sodium citrate and the clot is extremely weak.

By immunoenzymological assay, no release of fibrinopeptide A was noted after thrombin addition to purified fibrinogen. Furthermore, by disc electrophoresis in polyacrylamide gel in absence of SDS, an abnormal migration of the A α chain was observed.

These results led us to analyze the sequence of the N terminal part of the A α chain by the Edman procedure. Arginine in position 16 was replaced by cysteine in fibrinogen Metz. This mutation localized on the site cleaved by thrombin explains the absence of fibrinopeptide A release, and the abnormal clottability of fibrinogen Metz.
Abnormal Fibrin Monomer Polymerization In Patients With Lupus Erythematosus Systemicus (LES)
Authors: Nicastro M A , Molina M M , Acciaresi M A , Rios M Castro , Riveros D , Giavedoni E , Molinas F C
Published: July 2018

Fibrin monomer polymerization was studied in 10 out of 44 patients with LES, selected for their prolonged thrombin time (patients 24.6 ± 1.9 sec; normal controls 15.0 ± 0.5 sec). One of these patients had also a lupus-like blood clotting inhibitor. The level of fibrinogen measured as thrombin clottable protein was 407.5 ± 62.7 mg/dl, and as heat precipitable protein 325.8 ± 49.3 mg/dl. Fibrin monomer polymerization with thrombin assayed as the increment in O.D., showed in 5 patients a delay of two minutes compared to the normal controls. Under the same condition the O.D. did not show any change in 2 other patients. Purified IgG obtained from plasma of these patients had an inhibitory effect on the polymerization of human and bovine fibrinogen. IgG from normal plasma had no effect in this system. The fibrinogen of these patients had more anodic mobility than normal fibrinogen when studied by two-dimentional immunelectrophoresis. Factor XIII activity, euglobulin lysis time, and serum FDP gave results within normal range. We conclude that the prolonged thrombin time observed in some patients with LES seems to be the consequence of an abnormal fibrinogen function related to an interference by their own IgG.
Structure Of Fibrin
Authors: Freyssinet J M , Hudry-Clergeon G , Torbet J
Published: July 2018

Highly oriented fibrin gels are formed when polymerization takes place into a strong magnetic field-up to 200,000 G. Neutron low-angle diffraction show that the protofibrils pack into a three-dimensional crystalline lattice.

Kinetics parameters of the polymerization process can be estimated by recording the magnetically induced birefringence and the degree of orientation can be determined by measuring the final birefringence. To allow almost 100 % orientation, protein concentration has to be kept below 20 mg/ml (2 % w/v). Under these conditions X-ray low-angle diffraction patterns only show the familiar 225 Å-spaced meridional reflections. Neutron low-angle diffraction patterns are much more elaborated. In addition to the 223 Å (± 5 Å)-spaced meridional reflections, there are equatorial and other Bragg reflections which indicate that the fibrin monomers are arranged in a three-dimensional lattice. Meridional reflections due to a fibre axial periodicity are sharp, thus indicating that there is a high degree of crystallinity along the fibre axis. The equatorial diffraction arises from the regular, lateral arrangement of the fibrin monomers. In the pattern there is a discrete equatorial peak at 184 ± 5 Å and an extended band with a maximum at about 184/51/2 Å favouring a unit cell with a square base of side about 184 Å. Within each quadrant between the equator and the meridian, there are two maxima. The stronger of these is situated at the intersection of the first layer and row lines. The second is not so well resolved and has a meridional position between the second and third layer lines. So the true axial repeat might be 446 Å which agrees with the prevalent view that the approximately 450 Å-long molecules form a half-staggered arrangement along the fibre axis. Therefore this analysis, which is partly qualified, shows that the diffraction pattern can be indexed on a tetragonal unit cell (with a = 2 × 184 A and c =446 Å) that contains 8 molecules. Space groups P42 and P4222 satisfy our experimental conditions.
Laser Raman Spectroscopic Study Of The β-Sheet Structure Of Fibrinx
Authors: Marx J , Hudry-Clergeon G , Bernard L
Published: July 2018

Raman spectroscopy has proved to be a useful tool in the study of protein conformation in aqueous solution. Structural changes have been observed by this technique during the enzymatic conversion of fibrinogen into fibrin (J. Marx and col. (1979) Biochim. Biophys. Acta., 578, 107-115), particularly by the study of the Amide I and Amide III regions an important increase in the β-sheet form has been shown to occur. This variation is investigated under various conditions of ionic strength (μ) and protein concentration (c), two parameters which are known to change the fibre diameter (low values of μ and c favor an increase in the fibre diameter). The amount of β-sheet structure in fibrinogen is approximately 10 % and is unaffected by μ or c. In fibrin, the amount of β-sheet increases progressively from 20 % in fine clots (low diameter fibres) to more than 30 % in coarse clots (high diameter fibres). This correlation between the percentage of β-sheet structure and fibre diameter in fibrin indicates that numerous intermonomer hydrogen bonds are formed in the equatorial direction of the fibre. These bonds would greatly consolidate the association between monomers which is probably initiated at a few highly specific sites.
Lectin Binding Demonstrates Different Glycoprotein Abnormalities In Thrombasthenic And Bernard-Soulier Platelets
Authors: Judson Philip A , Anstee David J
Published: July 2018

We have previously shown that the lectins from Maclura aurantiaca (Ma) and Arachis hypogaea (Ah) (peanut) bind selectively to GP-I in human platelet membranes. In contrast Concanavalin A lectin binds primarily to GP-III. GP-I is deficient or absent from the membranes of individuals with Bernard-Soulier Syndrome (B-S.S.) while GP-II and III are deficient or absent from Thrombasthenic (G.T.) platelets. We have investigated the binding of radioiodinated lectins to SDS polyacrylamide gels of whole platelets from patients with B-S.S. and G.T. The object of this study was to define further the nature of the glycoprotein abnormalities in these conditions.

The results clearly demonstrated a marked reduction in the binding of Ma and Ah lectins to the GP-I region of B-S platelets when compared to that of normal platelets. No new lectin binding components were observed. The binding of Con A lectins to B-S platelets did not appear to be significantly different from that to normal platelets. In contrast Ma and Ah binding to G.T. platelets appeared normal whereas the binding of Con A to GP-III was markedly reduced. Two faint Con A binding bands were apparent in the GP-III region of the G.T. platelets. It is not clear whether either of these represent residual GP-III or if they are minor components masked by GP-III in normal platelets. The carbohydrate of GP-I in normal platelets consists almost entirely of O-glycosidically linked oligosaccharides. Ah and Ma lectins bind selectively to galactosyl and N-acetylgalactosaminyl residues in this type of oligosaccharide. We conclude that B-S platelets have a gross deficiency of O-glycosidically linked oligosaccharides. Con A binds selectively to branched N-glycosidically linked oligosaccharides with a mannose-rich core. We conclude that such structures normally present on GP-III are grossly deficient in G.T. platelets.
Membrane Glycoprotein Defects In Bernard-Soulier Syndrome Platelets
Authors: Clemetson K J , McGregor J L , James E , Dechavanne M , Lüscher E F
Published: July 2018

It is well established that Bernard-Soulier syndrome (BSS) platelets are deficient in a major membrane glycoprotein (lb). In order to investigate if this is the only defect in this disorder and to see if the β-subunit of glycoprotein lb is also diminished, platelets from 3 BSS patients and from healthy donors were isolated, washed and surface labelled by lactoperoxidase-catalysed iodination, pèriodate/NaB3H4 or neuraminidase/ galactose oxidase/NaB3H4. Labelled platelets were solubilized in sodium dodecyl sulphate and separated by 2-dimensional gel electrophoresis (isoelectric focusing, discontinuous polyacrylamide gel electrophoresis). Glycoprotein Ibα was virtually absent in 2 patients and strongly decreased in the third patient. The 3-subunit was also absent in the 2 patients and present at about 40 % of normal in the third. Glycoprotein IIbβ was present normally in all patients. In addition, a further low molecular weight glycoprotein with a M.WT. of 17,000 and a pI of 6.8-7.5 was absent or present at levels paralleling glycoprotein Ibβ. The thrombin cleavable glycoprotein (GP IV or V) appeared greatly diminished with BSS platelets labelled by carbo-hydrate specific methods though no difference could be seen with iodination. This finding was confirmed in a fourth BSS patient using one dimensional gel electrophoresis.

The defects in BSS platelets are thus more complex than previously thought.
Further Characterization Of Platelet Membrane Glycoproteins
Authors: Clezardin P , McGregor J L , Clemetson K J , Dechavanne M , Lüscher E F
Published: July 2018

The binding of 125I-labelled lectins to major and minor platelet glycoproteins (GP) and their subunits has been investigated. Human platelets were isolated, washed, solubilized in sodium dodecyl sulphate (SDS) under non-reducing conditions and separated on 5, 7.5 and 10 % non-reduced/reduced 2-D polyacrylamide gels. The gels were incubated with 125I-labelled lectins; Lens culinaris lectin (LCL), concanavalin A (ConA) wheat germ agglutinin (WGA) or Ricinus communis agglutinin (RCA-120), then washed extensively dried and exposed to X-ray film by indirect autoradiography. Surface-labelled platelets were similarly separated. WGA and RCA bound predominantly to GPIbα but also to two minor bands above and below it which were affected by neuraminidase treatment. One of them bound two 125I-lectins (LCL and ConA) while GPIbα did not. Additional GP bands were detected by lectin binding and by surface-labelling beneath GPIIIβ (IV). With platelets labelled by the neuraminidase/galactose oxidase/NaB3H4 method a GP was detected between Ila and Ilia which was not found with periodate/ NaB3H4 labelling (not affected by reduction). Two spots on the diagonal bound LCL and ConA. GP Ibβ bound LCL more strongly than IIbp. GPIbp also bound WGA and RCA. GPIcβ apparently bound only ConA. GPIbβ and IIbβ were labelled equally strongly by surface labelling techniques, Icβ was apparently not labelled. Further GP subunits were detected one below Ibβ and IIbβ and another which originated in the GPVII region. These techniques demonstrate that the platelet surface is even more complex than previously thought.
Montreal Platelet Syndrome (MPS):Ultrastructural, Biochemical And Functional Studies
Authors: Frojmovie M M , Milton J G , Tang S S
Published: July 2018

MPS is characterized by autosomal dominant inheritance thrombocytopenia (5,000-120,000 μl-1), abnormally large platelets on peripheral blood smear, spontaneous aggregation (SA), normal clot retraction and thromboplastin formation, nypervolumetric shape change and normal amounts of plasma membrane. Here we describe further characteristics of MPS for 3 siblings /5 affected family members. The ultrastructural features of MPS platelets were normal except for the high frequency of giant granules. Platelet aggregation (PA) was studied in citrated platelet-rich plasma at platelet counts of 5,000-40,000 μl-1 and was quantitated microscopically from the decrease in single platelet count. PA could be increased over that due to SA alone by the addition of ADP, adrenalin, collagen, ionophore A23187, arachidonic acid and ristocetin, suggesting that the response to these agents may be normal. The ristocetin-induced increase in PA was completely blocked by an IgG specific for BSS. In contrast, over a 4 year period, thrombin (0.2 units/ml) either did not or only slightly increased PA over SA for 3/3 donors. However, MPS platelets pelleted from ACD-PRP and resuspended in Tyrodes’, pH 7.4 at a platelet count of ~ 200,000 μ1-1 showed a normal response to thrombin by aggregometry. Only one donor’s platelets had both reduced sialic acid and glycoprotein (G.P.) 1 reduction/abnormality with otherwise normal G.P. and protein profile, while the other 2 siblings’ platelets showed no such abnormalities. The above results indicate that biochemical variability exists for MPS platelets from different affected siblings. Moreover, our observations raise the possibility that platelet aggregation abnormalities in thrombobytopenic disorders (may be obscured by (1) preparation artefacts and/or (2) artificially increasing platelet counts for in vitro studies.
Effect Of Moroxydinechloride, A Phenformin Like Substance, On Fibrinolysis In The Vessel Walls
Authors: Hender U , Tengborn L , Nilsson I M
Published: July 2018

It is well known that ethylestrenol and/or phenformin can normalise deficient fibrinolysis in the vessel walls and prevent recurrency of thromboembolism (Nilsson et al 1975, Hedner et al 1976). Because of the side-effects of these drugs we have studied the effect of a phenformin- like substance: moroxydinechloride (Kabi 1886) which does not cause lactic acidosis. Sixty-four patients with decreased fibrinolytic activity in vessel walls (histo- chemical assay of superficial hand vein according to Pan- dolfi et al 1971) and/or decreased release mechanism (venous occlusion test for 20 min as described by Robertson et al 1972) on repeated investigations are taking part in a prospective randomised clinical trial in which they receive Kabi 1886 in a dose of 0.04 g/kg and day or no specific treatment. All of these patients have earlier had one or more deep venous or arterial thrombosis or superficial thrombophlebitis. At check up 6 months after entering the trial it was found that 15 of 23 patients (65 %) with deficient release mechanism were normalised. Spontaneous normalisation in the control group was found in 5 of 18 patients (28 %). Some of the patients have been investigated also after 12 months and the tendency is the same as after 6 months. Treatment with Kabi 1886 thus seems to be effective in a fairly high percentage of patients with deficient release mechanism after venous occlusion.
Hypoxia And Acidosis Induce The Release Of Plasminogen Activator From Vessel Walls
Authors: Tappy L , Bachmann F
Published: July 2018

The mechanisms, which induce the release of vascular plasminogen activator (VA), are not fully understood. To test the effects of hypoxia and of acidosis on the liberation of VA fran the vessel wall, we have used the isolated pig ear perfusion system described by Markwardt and Klöcking. Pig ears, collected at the slaughterhouse, were kept on ice for a maximum of 4 hours. A catheter was placed into the main artery and the ear flushed with oxygenated HEPES-Tyrode’s solution pH 7,4 for 30 min at 37 °C. Baseline activities in effluent fractions from time 30-42 min, averaged 0,95 U UK/ml. Assays were performed on plasminogen rich fibrin plates. Frcm 43 to 65 min, pig ears were perfused with oxygenated HEPES-Tyrode's solution pH 7,4 (controls) or pH 6,7 (acidosis), or within the same buffer (pH 7,4) pretreated by extensive nitrogen bubbling (hypoxia).

VA concentrations in controls remained fairly constant until 65 min. The hypoxia stimulus resulted in an increase of PA activity of 180% over basal activity (p < 0,001). Acidosis led to a mean increase of PA activity of 240%
(p < 0,001)

These experiments suggest that acidosis and hypoxia, if present distally to venous segments occluded by a thrombus, may represent powerful stimuli for the secretion of plasminogen activator frcm vessel walls.
Fibrinolysis After Multiple Trauma In Man. Clinical And Methodological Aspects
Authors: Teger-Nilsson A-C , Gyzander E , Medegård A , Risberq B , Wiman B
Published: July 2018

Fifteen patients with at least two bone fractures and damages of soft tissues, mainly after traffic accidents, were investigated for changes in the fibrinolytic system. Seventeen healthy students were controls. The patients were conventionally treated in an intensive care unit. Blood samples, in all 140, were taken on the day of admission and then daily for about one week. The blood was analysed for plasminogen with electroimmunoassay and with chromogenic substrate after streptokinase activation, for α 2-antiplasmin with electroimmunoassay and two types of activity measurements (chromogenic substrate), for α 2-antiplasmin - plasmin complexes with RIA technique, and for α 2-macroglobulin with electroimmunoassay and activity measurements. In addition fibrinogen, fibrinogen-fibrin degradation products and fibrinolytic activity on fibrin plates were estimated.

Plasminogen was somewhat low the first days after trauma lowest 69 % ± 22 % (M ± SD), day 3]. It then slowly increased towards the end of the investigation (highest 123% ± 21%). The last days of investigation the immunological method also gave higher values than the activity method, α 2 Antiplasmin was low directly after the trauma (lowest 67% ± 18%, day 1 ) and increased gradually (highest 115% ± 19%, day 6 ). The first days after trauma immunological method gave higher values than the activity methods, α 2 Antiplasmin - plasmin complexes varied, but showed occasionally high values in particular the first day. α 2 -Macroglobulin decreased only slightly on day 5-6, when it also showed a difference between the immunological and activity methods.

It is concluded that the fibrinolytic system is activated immediately after a trauma. Later there is an increase of plasminogen, α 2 antiplasmin and fibrinogen, probably due to an acute phase reaction. Some reasons for the methodological discrepancies are discussed. Serial analysis of α 2-antiplasmin activity seems to be a relevant method for evaluation of fibrinolysis in a complex clinical situation.