Phosphorylation of factor Va and factor VIIIa by activated platelets

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.     

Assessment of obstruction in adult ureterocele by means of color Doppler duplex sonography

AIM: The purpose of this study is to present a method for identifying a ureteral obstruction in unilateral orthotopic ureterocele by means of conventional sonography and color Doppler duplex sonography. We focus on the measurement of the ureterocele dimensions, the degree of dilation it causes to the ipsilateral upper urinary tract, the registration of urine out-flow from the ureteral orifice into the bladder and its spectral analysis. MATERIAL AND METHOD: Over 2 years at our institutions, 8 adult patients (7 women, 1 man) were diagnosed as having a single system orthotopic ureterocele. Four of them presented with lumbar pain, dysuria and recurrent urinary tract infections, while the remainder were asymptomatic and diagnosed accidentally. The diagnosis was based on serial sonography of the upper and lower urinary tract confirmed by intravenous pyelography and cystoscopy. We also performed color Doppler duplex sonographic evaluation of the urine jets ejected from both ureteral orifices into the bladder. Using the flow spectral study we analyzed the waveforms and measured their duration and flow rate. The study was completed with a comparative analysis of the data obtained from both ureteral orifices. RESULTS: Cystic dilation of the lower ureteric extremity into the bladder was presented in all cases. Upper urinary tract dilation, of various grades, was present in 4 of 8 patients. Differences in urine jets between those derived from the ureterocele and those from the healthy contralateral ureteral orifice were significant in those patients with dilation of the upper urinary tract. The differences concerned mainly the frequency and symmetry of the jets as well as the pattern, duration and velocity of their waves. The 4 above-mentioned patients, with dilated upper urinary tracts and waveforms differentiated from the contralateral ones, were characterized as obstructive. On the other hand, the remaining 4 patients with subclinical ureterocele showed insignificant differences in urine jets and waveforms, and were found to be non-obstructive. CONCLUSION: Conventional sonography of the urinary tract in combination with color Doppler duplex sonography of the ureteral jets can be used in an attempt to diagnose and evaluate a unilateral orthotopic (single system) ureterocele and assess the necessity of intervention to identify the obstruction.     

Nephrogenic adenoma of the urinary bladder

Introduction: The nephrogenic adenoma (NA) is a benign metaplastic lesion of the urothelium and is attributed to chronic irritation of the mucosa, by injury, infection, stone disease or intravesical instrumentations. We present our experience on this morbid entity, its clinical appearance in the urinary bladder, its frequency and relapses. Furthermore we reviewed the related recent literature and focused on its potential to neoplastic degeneration and the value of the new diagnostic modalities. Patients and methods: Four patients with NA of the urinary bladder are presented. The papilloid or polypoid formations observed by the cystoscopy were identified after the TUR, as NA of the urinary bladder. Their mean follow-up was 3.5 years. Results: Remission of the symptoms was observed after TUR in all patients. Three out of four patients presented 1–7 relapses, while in one case, after seven NA relapses, a urothelial carcinoma of the bladder was diagnosed. Conclusions: Unlike histological features, the clinical – endoscopic characteristics of NA are non-specific. Even if it is not definitely considered like a premalignant condition, NA has to be followed up frequently and long lasting, because of its high recurrence rate. The combination of Cytology, Flow cytometry, DNA image analysis and Fluorescence in situ hybridisation of bladder washings or voided urine, are of high value in monitoring NA of the urothelium.     

Evaluation of capsule endoscopy in active, mild-to-moderate, overt, obscure GI bleeding

BACKGROUND: The role of capsule endoscopy (CE) in the diagnosis of active mild-to-moderate GI bleeding (GIB) immediately after a negative EGD and ileocolonoscopy has not been prospectively evaluated. OBJECTIVE: To estimate the diagnostic yield and clinical significance of CE in patients with acute, obscure, overt, mild-to-moderate GIB. DESIGN: A single-center prospective study. PATIENTS: During a 3-year period, 573 patients admitted to the hospital with acute mild-to-moderate GIB were included in this study. Among them, 37 patients (6.5%) with negative endoscopic findings, after urgent upper- and lower-GI endoscopies, underwent CE within the first 48 hours to identify the source of bleeding. RESULTS: CE revealed active bleeding in 34 patients and a diagnostic yield of 91.9%, including angiodysplasias in 18 patients, ulcers in 3 patients, and tumors in 2 patients. In the remaining 11 patients (32%), CE revealed the site of bleeding: distal duodenum in 1 case (9%), jejunum in 6 cases (54%), ileum in 2 cases (18%), and cecum in 2 cases (18%). From the 37 bleeders, 16 were managed conservatively, 14 endoscopically, and 7 surgically. During a 12-month follow-up period, bleeding recurrence was observed in 5 of 32 (15.6%). LIMITATIONS: This study had a limited number of patients. CONCLUSIONS: CE appeared to have a high diagnostic yield in patients with acute, mild-to-moderate, active hemorrhage of obscure origin when performed in the hospital after a negative standard endoscopic evaluation and has important clinical value in guiding medical management.     

Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (protein C(R-1L/propeptide)).

A heterozygous G-->T transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.     

Abnormality of the N-terminal portion of von Willebrand factor in type IIA and IIC von Willebrand disease

We have established a new analytical method which allows the characterization of von Willebrand factor (vWF) degradation fragments in minute amounts (10 microliters) of plasma, without the need for immunopurification of vWF. Plasma vWF was hydrolysed by S aureus V-8 protease (V-8 protease) and the cleaved fragments separated by SDS-agarose gel electrophoresis followed by staining with 125I-labeled polyclonal or monoclonal antibodies against vWF and autoradiography. Quantification of the amount of each product was estimated by counting the incorporated radioactivity following excision. V-8 protease limitedly hydrolysed vWF in normal as well as type I von Willebrand disease (vWD) plasma and produced two distinct fragments with identical electrophoretic and antigenic characteristics to those produced from purified vWF, i.e. a C-terminal SpII and a series of N-terminal SpIII fragments (SpIIIa, b and c). The method was applied to further characterize the molecular abnormalities of vWF in eighteen patients with type II vWD. In seven individuals with type IIA and five patients with type IIC, SpIII appeared significantly modified as compared to normal. In type IIA, there was a marked decrease or absence of SpIIIa and an increase of SpIIIb and c. In type IIC, SpIIIb was lacking. In three patients with type IIB and in three patients with type IID, there was no significant modification of SpIII. In all cases, SpII was apparently not modified. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in SpIII, the N-terminal portion of the vWF-subunit (residues 1 to 1,365).     

Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.     

Coagulation factor V: a plethora of anticoagulant molecules

PURPOSE OF REVIEW: Thrombin is necessary for survival and is produced after activation of prothrombin by prothrombinase at the site of a vascular injury. While the enzyme component of prothrombinase alone, factor Xa, bound to a membrane surface can activate prothrombin, incorporation of the cofactor molecule, factor Va, into prothrombinase results in a five orders of magnitude increase in the catalytic efficiency of factor Xa that provides the physiologic pathway for thrombin generation. While the kinetic constants and the identity of peptide bonds cleaved in prothrombin to generate alpha-thrombin have been long established, the peptidyl portions of the factor Va molecule responsible for its interactions with factor Xa, prothrombin, and the lipid surface are still the subject of intense investigation. In this review, we summarize the current state of knowledge with respect to the interactions of the factor Va molecule with the various components of prothrombinase. RECENT FINDINGS: Binding sites for factor Xa have been identified on both the heavy and light chains of factor Va. Two amino acid regions that interact with factor Xa have been delineated on the heavy chain of the cofactor. It has also been demonstrated that the carboxyl-terminal portion of the heavy chain of factor Va contains hirudin-like motifs and appears to be responsible for the interaction of factor Va with prothrombin. This region of the molecule is important for procofactor activation by thrombin as well as cofactor function. Finally, the membrane-binding site of factor Va is contributed by several elements of the light chain and involves both electrostatic and hydrophobic interactions. SUMMARY: The absence or dysfunction of factor Va leads to hemorrhagic diseases while prolonged existence of the active cofactor species is associated with thrombosis. Thus, modulation of the incorporation of factor Va into prothrombinase in vivo by using synthetic peptides that have the potential to impair factor Va binding to any of the components of prothrombinase, will allow for control of the rate of thrombin generation at the site of vascular damage. As a consequence, a systematic definition of the regions of factor Va governing its incorporation within prothrombinase will provide the scaffold for the synthesis of potent anticoagulant molecules that could modulate thrombin formation and suppress excessive clotting in thrombotic individuals.     

Adenomatous polyp of the verumontanum accompanied by parabulbous epidermoid cyst and cryptorchidism in an adult male

The coexistence of polyp of the verumontanum, epidermoid paraurethral cyst and cryptorchidism presents a mosaic of congenital anomalies of the urogenital tract. We present a 30-year-old patient with a medical history of unilateral cryptorchidism, urinary tract infections, obstructive voiding symptoms and perineal discomfort. Clinical and laboratory evaluation revealed a pending filling defect in the prostatic urethra and a palpable elastic mass located on the right side of the bulbar urethra. Transurethral resection of the urethral polyp with a simultaneous excision of the cystic formation via a perineal approach followed by a left-sided funiculolysis and orchiopexy were performed. Discussion focuses on the etiology and differential diagnosis of this unusual complex entity.