Evidence-based medicine: Rhinoplasty.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Discuss the key components in the aesthetic, functional, and emotional evaluation of a patient presenting for rhinoplasty. 2. Describe the various surgical options to achieve the goals identified during consultation. 3. Recognize appropriate nonsurgical options and their limitations for treating nasal deformities. 4. Assess the benefits and risks of using perioperative antibiotics in rhinoplasty. 5. Identify common postoperative sequelae of rhinoplasty and formulate a plan of care based on best evidence data. SUMMARY: This article was prepared to accompany practice-based assessment with ongoing surgical education for the Maintenance of Certification for the American Board of Plastic Surgery. It outlines the evaluation of the rhinoplasty patient and highlights both surgical and nonsurgical options to address nasal defects. In addition, data from the current literature are included to supplement physician knowledge for enhanced perioperative care and improved outcomes.

Enhancing lipoaspirate efficiency by altering liposuction cannula design.

BACKGROUND: Interplay between the components of a lipoplasty system (suction pump, suction tubing, collection canister, and cannula) determines liposuction efficiency. However, in clinical practice, none of the components are more important than the cannula. Cannula design including port design, port placement, and shaft characteristics is the single most influential contributor to flow resistance and dramatically effects speed of aspiration and final contour. Many variations on port design and placement are available, yet functional enhancements to the cannula shaft have largely been ignored. We have engineered a set of novel cannulas addressing vital elements of cannula design in the effort to enhance aspiration efficiency and efficacy. METHODS: Two novel cannula designs (dual- and multiport, in-line configuration), created using a unique proprietary manufacturing process, were evaluated against a popular industry standard design (tri-port, Mercedes configuration) to assess aspiration efficiency. Cannulas with shaft diameters of 3, 4, and 5 mm were attached to a standardized lipoplasty system and evaluated in real time for their ability to aspirate a viscous applesauce medium over a 5-minute time course. For each cannula, we calculated (1) the cross-sectional area of the cannula shaft, (2) single and total port area, (3) port-to-shaft ratio, and (4) theoretical resistance. RESULTS: The relationship between the cannula shaft and cannula port(s) directly influenced flow dynamics. Comparing medium uptake time, aspiration efficiency and the aspiration curves demonstrated a significant improvement of the 2 novel cannulas over the standard cannula in the 5- and 4-mm designations. In the 3-mm group, a difference in uptake time remained. However, a significant difference in aspiration efficiency was only seen between the dual-port novel cannula and tri-port Mercedes standard cannula. Further, differences in the aspiration curves between all 3-mm cannulas approached but did not reach significance. CONCLUSIONS: We have developed 2 novel cannulas that maximize port features and seek to minimize the internal shaft resistance. Both designs demonstrate enhanced aspiration and uptake compared with an industry standard design.

The presentation and management of hemangiomas.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: (1) Define what is meant by either a classic (infantile) or atypical hemangioma and understand the natural history of each. (2) Identify the common phenotypic, histologic, and radiographic findings of a hemangioma. (3) Know the potential complications associated with hemangiomas. (4) Outline the options available to treat a hemangioma. BACKGROUND: Hemangiomas represent the most common tumors of infancy. These lesions maintain unique patterns of growth, sequelae, and therapeutic modalities from other vascular birthmarks. Until recently, however, classification schemes based on historical phenotypic observations often complicated diagnosis and proper medical management. METHODS: Mulliken and Glowacki redefined the identification of these lesions by correlating clinical, histological, and autoradiographical data from a series of patients with vascular marks. A formal classification was officially adopted by the International Society for the Study of Vascular Anomalies in 1996 to structure a clinically relevant classification system. RESULTS: Under the adopted parameters, hemangiomas are classified as a subset of vascular tumors and delineated from vascular malformations. Generally, a hemangioma experiences a phase of rapid growth and expansion followed by slow, but steady, regression. The life cycle can be divided into the proliferating phase, involuting phase, and involuted phase, with each distinct in its time course and histological, radiographic, and molecular findings. Rarely, hemangiomas with an atypical presentation arise, and the clinician must be aware of their existence to avoid incorrect intervention and to prepare for possible life-threatening sequelae. CONCLUSIONS: The frequency of vascular marks in the general population makes it likely that a plastic surgeon will be involved in treatment of an affected patient at some point in his or her career. The goal of this article is to distinguish hemangiomas from other vascular marks by highlighting their presentation, possible complications, and common treatment modalities to aid diagnosis and therapeutic planning for this common vascular tumor.

Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII.

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

The contribution of amino acid region ASP695-TYR698 of factor V to procofactor activation and factor Va function.

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --> Lys, Tyr(696) --> Phe, Asp(697) --> Lys, and Tyr(698) --> Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

Structural requirements for expression of factor Va activity.

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.

Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity.

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --> Phe/Tyr324 --> Phe, factor VaFF; Glu330 --> Met/Val331 --> Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --> Phe/Tyr324 --> Phe and Glu330 --> Met/Val331 --> Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity.

Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X.

We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.

Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations.

A 44-year-old woman with a history of severe thrombotic manifestations presented with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA sequencing of and around the regions encoding the APC cleavage sites in the factor Va molecule excluded the presence of the factor VLeiden mutation and of other known genetic mutations. No antiphospholipid antibodies were present in the patient's plasma and both prothrombin time and activated partial thromboplastin time were normal. The total immunoglobulin fraction was isolated from the patient's plasma and found to induce severe APC resistance when added to normal plasma and to factor V-deficient plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total immunoglobulin fraction purified from the patient's plasma demonstrated that the antibody recognizes factor V, is polyclonal, and has conformational epitopes on the entire factor V molecule (heavy and light chains, and B region). Thus, the immunoglobulin fraction interferes with the anticoagulant pathway involving factor V. The inhibitor was isolated by sequential affinity chromatography on protein G-Sepharose and factor V-Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The isolated immunoglobulin fraction was also found to inhibit the cofactor effect of factor V for the inactivation of factor VIII by the APC/protein S complex. Our data provide for the first time the demonstration of an antifactor V antibody not related to the presence of antiphospholipid antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.