Impact of a surgical site infection reduction strategy after colorectal resection.

AIM: This study was performed to determine the impact of a surgical site infection (SSI) reduction strategy on SSI rates following colorectal resection. METHOD: American College of Surgeons National Surgical Quality Improvement Program (NSQIP) data from 2006-14 were utilized and supplemented by institutional review board-approved chart review. The primary end-point was superficial and deep incisional SSI. The inclusion criterion was colorectal resection. The SSI reduction strategy consisted of preoperative (blood glucose, bowel preparation, shower, hair removal), intra-operative (prophylactic antibiotics, antimicrobial incisional drape, wound protector, wound closure technique) and postoperative (wound dressing technique) components. The SSI reduction strategy was prospectively implemented and compared with historical controls (pre-SSI strategy arm). Statistical analysis included Pearson's chi-square test, and Student's t-test performed with spss software. RESULTS: Of 1018 patients, 379 were in the pre-SSI strategy arm, 311 in the SSI strategy arm and 328 were included to test durability. The study arms were comparable for all measured parameters. Preoperative wound class, operation time, resection type and stoma creation did not differ significantly. The SSI strategy arm demonstrated a significant decrease in overall SSI rates (32.19% vs 18.97%) and superficial SSI rates (23.48% vs 8.04%). Deep SSI and organ space rates did not differ. A review of patients testing durability demonstrated continued improvement in overall SSI rates (8.23%). CONCLUSION: The implementation of an SSI reduction strategy resulted in a 41% decrease in SSI rates following colorectal resection over its initial 3 years, and its durability as demonstrated by continuing improvement was seen over an additional 2 years.

Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.

BACKGROUND: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. OBJECTIVE: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. METHODS: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. RESULTS: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). CONCLUSIONS: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.

P-selectin antagonism reduces thrombus formation in humans.

BACKGROUND: Interaction of P-selectin with its glycoprotein ligand (P-selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P-selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. AIM: To investigate the effects of the novel small molecule P-selectin antagonist PSI-697 on thrombus formation in humans. METHODS AND RESULTS: In a double-blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low-dose (2 m) and high-dose (20 m) PSI-697 and the glycoprotein IIb-IIIa receptor antagonist tirofiban (50 ng mL(-1)) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb-IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low-shear and high-shear chambers, respectively. PSI-697 caused a dose-dependent, but more modest, reduction in thrombus formation. Low-dose PSI-796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low-shear and high-shear chambers, respectively. At the high dose (20 m), PSI-697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low-shear and high-shear chambers, respectively. CONCLUSIONS: P-selectin antagonism with PSI-697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P-selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.

Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca2+ channels.

Non-peptidic small-molecule antagonists of the human platelet thrombin receptor PAR-1.

The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus is referred to as the Thrombin Receptor Activating Peptide, or "TRAP" Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.

Discovery and initial structure-activity relationships of trisubstituted ureas as thrombin receptor (PAR-1) antagonists.

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.

The structure of leech anti-platelet protein, an inhibitor of haemostasis.

Leech anti-platelet protein (LAPP) from the leech Haementeria officinalis is a collagen-binding protein that inhibits the collagen-mediated adhesion of blood platelets. The crystal structure of recombinant LAPP has been determined using single isomorphous replacement with anomalous scattering combined with solvent flattening and threefold molecular averaging. The model of LAPP has been refined to 2.2 A resolution (R factor 21.5%; free R factor 24.0%). LAPP contains an 89-residue C-terminal domain consisting of a central six-stranded antiparallel beta-sheet flanked on one side by an alpha-helix and on the other side by two extended loops with little secondary structure. A 36-residue N-terminal region is not visible in the electron-density map. This region is rich in glycine and lacks hydrophobic residues. It probably does not have a compact globular fold, but instead has an extended conformation and is flexible. The crystal packing suggests that LAPP may form tightly interacting dimers. The fold of the C-terminal domain of LAPP closely resembles that of the N-domain of hepatocyte growth factor (HGF), which classifies LAPP as a PAN domain. However, no significant sequence homology exists between LAPP and other PAN domains. Common structural features between LAPP and the HGF N-domain include two disulfide bonds that link the alpha-helix to the central region of the protein and five residues with a conserved hydrophobic nature that are located in the core of the domain. These conserved structural features may be an important determinant of the PAN-domain type of fold.

The interaction of disagregin with the platelet fibrinogen receptor, glycoprotein IIb-IIIa.

Disagregin, a 6 kDa protein isolated from salivary glands of the tick Ornithodoros Moubata, is a potent and selective inhibitor of fibrinogen dependent platelet aggregation and of the adhesion of platelets to fibrinogen (Karczewski et al. (1994) J. Biol. Chem. 269, 6702-6708). In the current study the interaction of disagregin with purified glycoprotein IIb-IIIa (GPIIb-IIIa) was examined. Biotin-labeled disagregin (b-disagregin) bound to GPIIb-IIIa immobilized on the surface of the ELISA plate. This binding was specific, dependent on divalent cations, and was blocked by the peptides fibrinogen gamma-chain fg gamma (400-411), GPIIb(296-306) and by the RGD-containing peptide, GRGDSP. Disagregin also bound to soluble GPIIb-IIIa as demonstrated in studies using the chemical crosslinker, BS3. This binding was inhibited by the peptides fg gamma (400-411) and GPIIb(296-306). In contrast to the results in the solid phase, peptide GRGDSP had no effect on the binding of b-disagregin to soluble GPIIb-IIIa. These data demonstrate that disagregin binds to GPIIb-IIIa through a mechanism distinct from that used by RGD-containing disintegrins. Further analysis of the region(s) of disagregin which bind to GPIIb-IIIa should provide useful information for molecular modeling of the fibrinogen binding site on GPIIb-IIIa and for the design of a new class of potent fibrinogen receptor antagonists.