Women and inherited bleeding disorders - A review with a focus on key challenges for 2019.

The area of women and inherited bleeding disorders has undergone quick expansion in recent years. More patients are being identified and expertise to diagnose and manage these patients is now essential for practising physicians. Programs to help educate and empower patients and caregivers are now in place. Common inherited bleeding disorders affecting women include von Willebrand disease (VWD), inherited platelet disorders, and rare inherited bleeding disorders such as factor VII (FVII) deficiency and factor XI (FXI) deficiency. Specific clinical tools have been developed to help clinicians and patients screen for the presence of these bleeding disorders in both adult and pediatric populations. Affected women can experience heavy menstrual bleeding and resulting iron deficiency anemia, postpartum hemorrhage, and hemorrhagic ovarian cysts which need to be properly managed. Excessive bleeding can adversely affect quality of life in these women. Front line therapy for bleeding in mild cases focuses on the use of non-specific hemostatic agents such as DDAVP ®, tranexamic acid and hormonal agents but specific factor replacement and/or blood products may be required in more severe cases, in severe bleeding or as second line treatment when bleeding is not responsive to first line agents. Iron status should be optimised in these women especially in pregnancy and use of an electronic app can now help clinicians achieve this. These patients should ideally be managed by a multidisciplinary team whenever possible even remotely. Although clinical research has closed some knowledge gaps regarding the diagnosis and management of these women, there remains significant variation in practise and lack of evidence-based guidelines still exists in many spheres of clinical care in which caregivers must rely on expert opinion. Ongoing efforts in education and research will continue to improve care for these women and restore quality of life for them.

Integrins in drug targeting-RGD templates in toxins.

Integrins are a family of heterodimeric receptors, which modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Integrin-binding RGD (arginine-glycine-aspartic acid) is found in several important extracellular matrix proteins which serve as adhesive integrin ligands. The RGD motif has also been found in many toxins from snake venom and other sources that specifically inhibit integrin binding function and serve as potent integrin antagonists, particularly of platelet aggregation and integrin-mediated cell adhesion. Many of these proteins have potential as therapeutic agents which can target integrins directly. Structural and functional studies of several RGD-containing toxins suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD at the tip of a flexible loop, a structural feature for binding to integrins. In addition, amino acid residues in this loop in close vicinity to the RGD-motif determine the integrin-binding specificity and selectivity. This review will present a review of integrin structure and function, and of disintegrin structural features responsible for their activity as antagonists of integrin function. The use of integrins in drug targeting and integrins as targets for drug delivery by using the RGD as a template structure will also be discussed.

Structural features of fibrinogen associated with binding to chelated zinc.

Fibrinogen absorbed to zinc chelate columns at pH 7.8 and was eluted sharply with a pK of 5.8 indicative of the involvement of a histidine residue. The binding was unaffected by high concentrations of calcium or zinc. Fibrinogen degradation products X and Y also bound to zinc chelate columns. On passage of fibrinogen fragments D and E through these columns fragment E was not bound and fragment D was eluted subsequently in a pure form with a pK of 6.1.

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.

Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins.

Snake venom disintegrins represent a family of RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp)-containing proteins which have been reported to be unique and potentially useful tools not only for investigating integrin-ligand interactions, but also for the development of anti-thrombotic agents in terms of their anti-platelet activities. Snake venom proteins containing a disintegrin-like domain represent another super-family of proteins in which many of them have been demonstrated to have similar ability to inhibit platelet aggregation and integrin-mediated cell adhesion as the disintegrins. This super-family includes a large number of snake venom metalloproteinases and disintegrin related, RGD-containing snake venom proteins (disintegrin-like proteins) such as dendroaspin. Recently, a family of homologues of the snake venom metalloproteinases have been found in a wide variety of mammalian tissues as well as in other eukaryotic organisms termed ADAM (a disintegrin-like and metalloproteinase) proteins. ADAMs are members of the metazincins that also include the related matrix metalloprotease (MMPs). Some of ADAM proteins have now shown to interact with integrins, and the disintegrin-like domain may be crucial part in their function as proteases. A description of structure-activity relationships of snake venom proteins containing a disintegrin-like domain is outlined in this review, along with reports of the modulation of protein activity by recombinant mutation. Comparison is also made of the structural and functional features of the metalloproteinases in snakes compared with those from other species. The review is intended to provide insights in which may assist the development of new therapeutic approaches.

Fibrin subunits in venous and arterial thromboembolism.

The subunit fibrin composition of thrombi of both venous and arterial origin was examined by sodium dodecyl sulphate gel electrophoresis. The thrombi were recovered by surgical intervention and all had the same fibrin subunit composition. The alpha chains were cross-linked as alpha-chain polymers alpha (p), the gamma chains as gamma-chain dimers (gamma-gamma) while the beta chains were not crosslinked; a further subunit of molecular weight 33 000 was shown to be present in all the fibrins examined and was a degradation fragment of the beta or gamma chains. This data suggests that the crosslinked alpha chains are rate limiting to the lysis of thrombi in vivo. The digestion of pulmonary emboli by plasmin yielded soluble degradation products which were identified as D dimer and E, the latter fragments being the major products obtained by the lysis of in-vitro made plasma clots. The similarity of the composition and lysis of thrombus fibrin to that formed in vitro augurs well for the justification of in-vitro research on mechanisms in thrombolysis.

Heparan sulphate with no affinity for antithrombin III and the control of haemostasis.

Heparan sulphate with no affinity for antithrombin III (ATIII) was observed to cause acceleration of the factor Xa:ATIII interaction by 1100-fold (k2, 7 X 10(7) M-1.min-1) and the prothrombinase:ATIII interaction by 2900-fold (k2, 2.5 X 10(7) M-1.min-1). Although high-affinity heparan sulphate catalyzed higher acceleration and at lower concentration, in natural mixtures of the two forms the activity of the no affinity form predominated. Heparan sulphate had no significant effect on the thrombin:ATIII interaction but inhibited its potentiation by heparin (Kd 0.3 microM). From the estimated concentration of heparan sulphate on the endothelial cell surface it is proposed that the non-thrombogenic property of blood vessels is due to the acceleration of the factor Xa or prothrombinase:ATIII interaction by the greater mass of surface-bound heparan sulphate rather than by the much smaller proportion of heparin-like molecules (with high affinity for antithrombin III) which may be present.

A cyclic peptide analogue of loop III of PDGF-BB causes apoptosis in human fibroblasts.

A cyclic peptide analogue of platelet-derived growth factor-BB (PDGF-BB), P1 [77IVRKK81-C-73RKIE76], has recently been shown to inhibit specifically [125I]PDGF-BB/receptor binding, and PDGF-BB-induced DNA synthesis in cells expressing PDGF receptors. Here we demonstrate that P1 induces apoptosis in exponentially growing human fibroblasts as confirmed by characteristic changes in cell and nuclear morphology, by TUNEL staining and by flow cytometry. Following incubation with P1 (100 microM), the percentage of cells exhibiting DNA fragmentation increased from 24% after 8 h to 76% after 28 h as exponentially growing cells progressed through the cell cycle. We conclude from these findings taken together that apoptosis accounts for the major proportion of P1-induced cell death. Omission of the Cys residue from P1 or replacement by Ser did not alter the potency of the peptide confirming that peptide dimerisation is not important for its activity. PDGF-BB, EGF, FGF, thrombin and foetal bovine serum were not able to rescue cells from the effects of P1. P1 is a useful tool for investigation of the balance of cellular proliferation/apoptosis in wound healing, atherosclerosis and restenosis, and constitutes a basis from which to design compounds with greater potency.

Identification of a cyclic peptide inhibitor of platelet-derived growth factor-BB receptor-binding and mitogen-induced DNA synthesis in human fibroblasts.

Peptides corresponding to residues from Loops I and III of platelet-derived growth factor-BB (PDGF-BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF-induced DNA synthesis. The inhibitory action of cyclic PDGF-BB(73-81), on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF-BB(73-81) specifically competed for 125I-labelled PDGF-BB but not for 125I-labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF-BB(73-81) in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.

Influence of intrinsic and extrinsic plasminogen upon the lysis of thrombi in vitro.

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

Influence of tryptophan modification upon digestion of antithrombin III by elastase.

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

High affinity binding of heparin by necrotic tumour cells neutralises anticoagulant activity--implications for cancer related thromboembolism and heparin therapy.

We have observed a striking neutralisation of the anticoagulant activity of unfractionated heparin in the presence of a pancreatic carcinoma cell line (MIA PaCa-2) due to binding of around 9 microg of heparin per 10(7) cells (apparent Kd, 30 nM). The loss of anticoagulant activity was less marked in the presence of low molecular weight forms of heparin. Binding to the cell blocked acceleration of the thrombin:antithrombin interaction by heparin. Neutralisation of heparin activity was also shown to occur in the presence of a number of other tumour cell lines. FACS analysis demonstrated that live cells did not bind heparin and high affinity binding only occurred to dead MIA PaCa-2 cells. Heparin binding proteins accumulating in cell medium were identified as histone and ribosomal proteins that will become exposed during necrosis. The release of these proteins from cells within the necrotic core of a tumour or from cells killed during chemotherapy may abrogate the heparan sulphate/antithrombin system and possibly contribute to the idiopathic thromboembolism often associated with cancer (Trousseau's syndrome). The findings also suggest a reason for the reported advantage of LMWH over UFH in treating venous thromboembolism in cancer patients and in improving patient survival.

Properties of thrombolytic agents in vitro using a perfusion circuit attaining shear stress at physiological levels.

A perfusion circuit was designed to investigate in vitro some of the factors which may influence the success of thrombolytic treatment in vivo. The rate of lysis of clotted plasma and different types of artificial thrombi (fibrin thrombi or whole blood thrombi) was measured in citrated plasma or whole blood under static conditions or under shear stress equivalent to the arterial or venous circulation. With both streptokinase (SK) and tissue-type plasminogen activator (t-PA) the rate of lysis of fibrin thrombi and whole blood thrombi was reduced significantly, when compared to the conventional plasma gel clot model (25-fold and 8-fold, respectively). This occurred particularly with SK which showed a reduction (4-fold) in potency relative to t-PA under these conditions. Lysis of thrombi by both activators was observed to be faster in plasma than whole blood, and also faster with whole blood thrombi than fibrin thrombi. High shear stress, generally, caused a reduction in the rate of lysis of fibrin thrombi and an increase in the rate of lysis of whole blood thrombi compared to lysis rates under static conditions. Under all conditions of flow the lysis rate observed at 50 units t-PA per ml was much faster than that at 500 units per ml unlike the conventional plasma gel clot model.

Influence of whole blood on standard curve for heparin measurement--possible heparin binding by red cells.

Measurement of heparin ex vivo is usually with reference to standard curve prepared with a "spiked" normal human plasma pool (NHP). When the calibration curve was prepared by addition of heparin to whole blood before plasma separation, although the linear relationship was maintained the slope was increased in comparison to the classical standard calibration curve. It is concluded that the preparation of the calibration curve by addition of heparin to NHP may give erroneously high heparin levels in treated patients' plasma, leading perhaps to inappropriate dosage. It was also observed that when heparin was added to blood of different haematocrit (prepared by addition of washed RBC to plasma) and plasma prepared, the subsequent APTT was decreased with the fall in haematocrit; suggesting that the laboratory monitoring of heparin treatment should take into account the patient's haematocrit.

Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator.

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of collagen mediated platelet release on plasma prekallikrein activation.

Collagen mediated platelet aggregation caused -5.6 +/- 6.7% inhibition and +39.1 +/- 15.2% potentiation of prekallikrein activation in plasma from normal healthy volunteers between 20-40 and 50-65 years of age, respectively (n = 15, p less than 0.01). The amounts of platelets factor-four (PF4) released in the two groups were not significantly different. Collagen treatment in the presence of indomethacin caused +11.5 +/- 3.6% and +59.6 +/- 19.5% potentiation in the 20-40 and 50-65 age groups respectively (p less than 0.02). Adrenaline mediated platelet aggregation caused -55.2 +/- 7.1% and -35.2 +/- 8.3% inhibition in the 20-40 and 50-65 age groups, respectively. Collagen treatment of platelet-deficient-plasma and platelet-rich-plasma in EDTA also caused potentiation of prekallikrein activation. The results indicate that the observed degree of prekallikrein activation after platelet aggregation is a net result of the inhibitory effect of PF4 and the potentiatory effect of activated platelets. The potentiatory effect was greater after collagen treatment as compared to adrenaline treatment, and in the 50-65 age group as compared to the 20-40 age group.

Intermittent plasminogen-streptokinase treatment of deep vein thrombosis.

The fibrinolytic response of 12 patients receiving single daily infusions of 600,000 units of streptokinase (SK) and 90 mg of plasminogen for the treatment of DVT has been studied. The mean plasminogen concentration was maintained throughout the treatment period (4-6 days) at between 20-40% the initial value, while mean circulating plasmin concentration rose to only about twice initial plasma levels. The degradation of fibrinogen as indicated by a fall in clottable fibrinogen did not fall below 1 mg/ml and serum FDP rose to greater than 1 mg/ml. Limited fibrinogenolysis occurred in 2 patients, while in another patient who bled there was immediate and extensive depletion to below 0.5 mg/ml. The beneficial clinical results obtained with this regimen (Kakkar et al. 1975), which produces only limited systemic plasminaemia, suggest that thrombolysis may be facilitated by higher levels of plasminogen than those maintained during conventional SK treatment.

Decrease in factor VII coagulant activity during percutaneous transluminal coronary angioplasty by heparin-mediated lipolytic action.

Levels of factor VII coagulant activity (FVII:C) and two-chain factor VIIa antigen (FVIIa:Ag) were measured in ten patients before and up to 6 h after receiving a bolus of heparin during percutaneous transluminal coronary angioplasty (PTCA). A significant and sustained post-heparin fall in the level of FVII:C was observed (approximately 30%) without any change in the level of FVIIa:Ag. The level of tissue factor antigen within the circulation remained unchanged. The observed decrease in FVII:C coincided with a significant decrease in triglyceride levels presumably due to lipoprotein and hepatic lipase released by the heparin. These findings appear to demonstrate a lipid (triglyceride) dependence of FVII:C. Thus, heparin may act indirectly as antithrombotic agent by limiting a lipid-dependent activation of the extrinsic pathway of coagulation.

The influence of DDAVP infusion on the coagulation and fibrinolytic response to surgery.

The response of components of the coagulation and fibrinolysis systems to infusion of DDAVP has been examined in patients undergoing elective surgery. In the DDAVP treated group there was a significant increase, compared to control, in plasminogen activator (by fibrin plates p less than 0.005, ECLT p less than 0.0125, by Student's t test) before operation. No difference between groups was seen by either methods in the activator levels in samples 24 h postoperation, whereas a significant drop (p less than 0.002) in protein C concentration was observed at this stage in the treated group. Levels of factor VIII components were significantly higher (p less than 0.005) than control at all stages of operation and a significant shortening (5 sec p less than 0.05) of the APTT was seen at all stages (apart from 24 h samples). DDAVP infusion therefore may exacerbate the hypercoagulable state observed in surgical patients without preventing the (post-operatively) fibrinolytic shutdown. Instead, infusion tends to produce fibrinolytic depletion at the key mid-operative stage.

The potential thrombogenic action of a nonionic radiographic contrast medium used during coronary angiography is offset by heparin during coronary angioplasty.

Iohexol sodium, a nonionic radiographic contrast medium, used in invasive imaging techniques has been shown to be potentially thrombogenic. In the present study, the effect of iohexol sodium on haemostatic factors was evaluated in 20 patients, 16 male and 4 female, 10 undergoing coronary angiography and another 10 undergoing coronary angioplasty. All the patients had angiographically-assessed coronary artery disease. The patients undergoing coronary angioplasty received a significantly larger quantity of the dye as compared with the patients undergoing coronary angiography. The former group of patients also received a bolus of 20,000 units of standard heparin in addition. The levels of thrombin-antithrombin-III complex (TAT), prothrombin fragments 1 and 2 (F1F2), D-dimer and the functional activity of tissue factor pathway inhibitor (TFPI) were assayed. While the baseline and 30-min post-dye levels of TAT and F1F2 were comparable in patients undergoing coronary angioplasty, the 30 min levels were significantly elevated in patients undergoing coronary angiography. The post-dye levels of TFPI activity were significantly increased in the former group due to the heparin-induced release of TFPI. It is concluded that the thrombogenic potential of iohexol sodium was overcome by heparin used routinely during coronary angioplasty.